Yeast microorganisms with reduced by-product accumulation for improved production of fuels, chemicals, and amino acids

ABSTRACT

The present invention relates to recombinant microorganisms comprising biosynthetic pathways and methods of using said recombinant microorganisms to produce various beneficial metabolites. In various aspects of the invention, the recombinant microorganisms may further comprise one or more modifications resulting in the reduction or elimination of 3 keto-acid (e.g., acetolactate and 2-aceto-2-hydroxybutyrate) and/or aldehyde-derived by-products. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the  Saccharomyces  Glade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/025,801, filed Feb. 11, 2011, which claims priority to U.S.Provisional Application Ser. No. 61/304,069, filed Feb. 12, 2010; U.S.Provisional Application Ser. No. 61/308,568, filed Feb. 26, 2010; U.S.Provisional Application Ser. No. 61/282,641, filed Mar. 10, 2010; U.S.Provisional Application Ser. No. 61/352,133, filed Jun. 7, 2010; U.S.Provisional Application Ser. No. 61/411,885, filed Nov. 9, 2010; andU.S. Provisional Application Ser. No. 61/430,801, filed Jan. 7, 2011,each of which is herein incorporated by reference in its entirety forall purposes.

ACKNOWLEDGMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Contract No.2009-10006-05919, awarded by the United States Department ofAgriculture, and under Contract No. W911 NF-09-2-0022, awarded by theUnited States Army Research Laboratory. The government has certainrights in the invention.

TECHNICAL FIELD

Recombinant microorganisms and methods of producing such organisms areprovided. Also provided are methods of producing beneficial metabolitesincluding fuels, chemicals, and amino acids by contacting a suitablesubstrate with recombinant microorganisms and enzymatic preparationstherefrom.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:GEVO_(—)045_(—)12US_SeqList_ST25.txt, date recorded: Sep. 18, 2013, filesize: 305 kilobytes).

BACKGROUND

The ability of microorganisms to convert pyruvate to beneficialmetabolites including fuels, chemicals, and amino acids has been widelydescribed in the literature in recent years. See, e.g., Alper et al.,2009, Nature Microbiol. Rev. 7: 715-723. Recombinant engineeringtechniques have enabled the creation of microorganisms that expressbiosynthetic pathways capable of producing a number of useful products,such as valine, isoleucine, leucine, and panthothenic acid (vitamin B5).In addition, fuels such as isobutanol have been produced recombinantlyin microorganisms expressing a heterologous metabolic pathway (See,e.g., WO/2007/050671 to Donaldson et al., and WO/2008/098227 to Liao, etal.). Although engineered microorganisms represent potentially usefultools for the renewable production of fuels, chemicals, and amino acids,many of these microorganisms have fallen short of commercial relevancedue to their low performance characteristics, including lowproductivity, low titers, and low yields.

One of the primary reasons for the sub-optimal performance observed inmany existing microorganisms is the undesirable conversion of pathwayintermediates to unwanted by-products. The present inventors haveidentified various by-products, including 2,3-dihydroxy-2-methylbutanoicacid (DH2MB) (CAS #14868-24-7), 2-ethyl-2,3-dihydroxybutyrate,2,3-dihydroxy-2-methyl-butanonate, isobutyrate, 3-methyl-1-butyrate,2-methyl-1-butyrate, and propionate, which are derived from variousintermediates of biosynthetic pathways used to produce fuels, chemicals,and amino acids. The accumulation of these by-products negativelyimpacts the synthesis and yield of desirable metabolites in a variety offermentation reactions. Until now, the enzymatic activities responsiblefor the production of these unwanted by-products had not beencharacterized. More particularly, the present application shows that theactivities of a 3-ketoacid reductase (3-KAR) and an aldehydedehydrogenase (ALDH) allow for the formation of these by-products fromimportant biosynthetic pathway intermediates.

The present invention results from the study of these enzymaticactivities and shows that the suppression of the 3-KAR and/or ALDHenzymes considerably reduces or eliminates the formation of unwantedby-products, and concomitantly improves the yields and titers ofbeneficial metabolites. The present application shows moreover, thatenhancement of the 3-KAR and/or ALDH enzymatic activities can be used toincrease the production of various by-products, such2,3-dihydroxy-2-methylbutanoic acid (DH2MB),2-ethyl-2,3-dihydroxybutyrate, 2,3-dihydroxy-2-methyl-butanonate,isobutyrate, 3-methyl-1-butyrate, 2-methyl-1-butyrate, and propionate.

SUMMARY OF THE INVENTION

The present inventors have discovered that unwanted by-products canaccumulate during various fermentation processes, including fermentationof the biofuel candidate, isobutanol. The accumulation of these unwantedby-products results from the undesirable conversion of pathwayintermediates including the 3-keto acids, acetolactate and2-aceto-2-hydroxybutyrate, and/or aldehydes, such as isobutyraldehyde,1-butanal, 1-propanal, 2-methyl-1-butanal, and 3-methyl-1-butanal. Theconversion of these intermediates to unwanted by-products can hinder theoptimal productivity and yield of a 3-keto acid- and/or aldehyde-derivedproducts. Therefore, the present inventors have developed methods forreducing the conversion of 3-keto acid and/or aldehyde intermediates tovarious fermentation by-products during processes where a 3-keto acidand/or an aldehyde acts as a pathway intermediate.

In a first aspect, the present invention relates to a recombinantmicroorganism comprising a biosynthetic pathway of which a 3-keto acidand/or an aldehyde is/are intermediate(s), wherein said recombinantmicroorganism is (a) substantially free of an enzyme catalyzing theconversion of a 3-keto acid to a 3-hydroxyacid; (b) substantially freeof an enzyme catalyzing the conversion of an aldehyde to an acidby-product; (c) engineered to reduce or eliminate the expression oractivity of an enzyme catalyzing the conversion of a 3-keto acid to a3-hydroxyacid; and/or (d) engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion of analdehyde to acid by-product. In one embodiment, the 3-keto acid isacetolactate. In another embodiment, the 3-keto acid is2-aceto-2-hydroxybutyrate.

In one embodiment, the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses the 3-ketoacid, acetolactate, as an intermediate, wherein said recombinantmicroorganism is engineered to reduce or eliminate the expression oractivity of an enzyme catalyzing the conversion of acetolactate to thecorresponding 3-hydroxyacid, DH2MB. In some embodiments, the enzymecatalyzing the conversion of acetolactate to DH2MB is a 3-ketoacidreductase (3-KAR).

In one embodiment, the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses the 3-ketoacid, 2-aceto-2-hydroxybutyrate, as an intermediate, wherein saidrecombinant microorganism is engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofacetolactate to the corresponding 3-hydroxyacid,2-ethyl-2,3-dihydroxybutanoate. In some embodiments, the enzymecatalyzing the conversion of 2-aceto-2-hydroxybutyrate to2-ethyl-2,3-dihydroxybutanoate is a 3-ketoacid reductase (3-KAR).

In one embodiment, the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses an aldehydeas an intermediate, wherein said recombinant microorganism is engineeredto reduce or eliminate the expression or activity of an enzymecatalyzing the conversion of the aldehyde to an acid by-product. In someembodiments, the enzyme catalyzing the conversion of the aldehyde to anacid by-product is an aldehyde dehydrogenase (ALDH).

In one embodiment, the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses both a 3-ketoacid and an aldehyde as intermediates, wherein said recombinantmicroorganism is (a) engineered to reduce or eliminate the expression oractivity of an enzyme catalyzing the conversion of a 3-keto acidintermediate to a 3-hydroxyacid by-product; and (b) engineered to reduceor eliminate the expression or activity of an enzyme catalyzing theconversion of an aldehyde intermediate to an acid by-product. In oneembodiment, the 3-keto acid is acetolactate and the 3-hydroxyacidby-product is DH2MB. In another embodiment, the 3-keto acid is2-aceto-2-hydroxybutyrate and the 3-hydroxyacid by-product is2-ethyl-2,3-dihydroxybutanoate. In some embodiments, the enzymecatalyzing the conversion of acetolactate to DH2MB is a 3-ketoacidreductase (3-KAR). In some other embodiments, the enzyme catalyzing theconversion of 2-aceto-2-hydroxybutyrate to2-ethyl-2,3-dihydroxybutanoate is a 3-ketoacid reductase (3-KAR). Insome other embodiments, the enzyme catalyzing the conversion of thealdehyde to an acid by-product is an aldehyde dehydrogenase (ALDH). Inyet some other embodiments, the enzyme catalyzing the conversion ofacetolactate to DH2MB is a 3-ketoacid reductase (3-KAR) and the enzymecatalyzing the conversion of the aldehyde to an acid by-product is analdehyde dehydrogenase (ALDH). In yet some other embodiments, the enzymecatalyzing the conversion of 2-aceto-2-hydroxybutyrate to2-ethyl-2,3-dihydroxybutanoate is a 3-ketoacid reductase (3-KAR) and theenzyme catalyzing the conversion of the aldehyde to an acid by-productis an aldehyde dehydrogenase (ALDH).

In various embodiments described herein, the recombinant microorganismsof the invention may comprise a reduction or deletion of the activity orexpression of one or more endogenous proteins involved in catalyzing theconversion of a 3-keto acid intermediate to a 3-hydroxyacid by-product.In one embodiment, the activity or expression of one or more endogenousproteins involved in catalyzing the conversion of a 3-keto acidintermediate to a 3-hydroxyacid by-product is reduced by at least about50%. In another embodiment, the activity or expression of one or moreendogenous proteins involved in catalyzing the conversion of a 3-ketoacid intermediate to a 3-hydroxyacid by-product is reduced by at leastabout 60%, by at least about 65%, by at least about 70%, by at leastabout 75%, by at least about 80%, by at least about 85%, by at leastabout 90%, by at least about 95%, or by at least about 99% as comparedto a recombinant microorganism not comprising a reduction or deletion ofthe activity or expression of one or more endogenous proteins involvedin catalyzing the conversion of a 3-keto acid intermediate to a3-hydroxyacid by-product. In one embodiment, the 3-keto acidintermediate is acetolactate and the 3-hydroxyacid by-product is DH2MB.In another embodiment, the 3-keto acid intermediate is2-aceto-2-hydroxybutyrate and the 3-hydroxyacid by-product is2-ethyl-2,3-dihydroxybutanoate.

In various embodiments described herein, the protein involved incatalyzing the conversion of a 3-keto acid intermediate to a3-hydroxyacid by-product is a ketoreductase. In an exemplary embodiment,the ketoreductase is a 3-ketoacid reductase (3-KAR). In anotherembodiment, the protein is a short chain alcohol dehydrogenase. In yetanother embodiment, the protein is a medium chain alcohol dehydrogenase.In yet another embodiment, the protein is an aldose reductase. In yetanother embodiment, the protein is a D-hydroxyacid dehydrogenase. In yetanother embodiment, the protein is a lactate dehydrogenase. In yetanother embodiment, the protein is selected from the group consisting ofYAL060W, YJR159W, YGL157W, YBL114W, YOR120W, YKL055C, YBR159W, YBR149W,YDL168W, YDR368W, YLR426W, YCR107W, YIL124W, YML054C, YOL151W, YMR318C,YMR226C, YBR046C, YHR104W, YIR036C, YDL174C, YDR541C, YBR145W, YGL039W,YCR105W, YDL124W, YIR035C, YFL056C, YNL274C, YLR255C, YGL185C, YGL256W,YJR096W, YMR226C, YJR155W, YPL275W, YOR388C, YLR070C, YMR083W, YER081W,YJR139C, YDL243C, YPL113C, YOL165C, YML086C, YMR303C, YDL246C, YLR070C,YHR063C, YNL331C, YFL057C, YIL155C, YOL086C, YAL061W, YDR127W, YPR127W,YCI018W, YIL074C, YIL124W, and YEL071W genes of S. cerevisiae andhomologs thereof.

In one embodiment, the endogenous protein is a 3-ketoacid reductase(3-KAR). In an exemplary embodiment, the 3-ketoacid reductase is the S.cerevisiae YMR226C (SEQ ID NO: 1) protein, used interchangeably hereinwith “TMA29”. In some embodiments, the endogenous protein may be the S.cerevisiae YMR226C (SEQ ID NO: 1) protein or a homolog or variantthereof. In one embodiment, the homolog may be selected from the groupconsisting of Vanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomycescastellii (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomycesbayanus (SEQ ID NO: 5), Zygosaccharomyces rouxii (SEQ ID NO: 6),Kluyveromyces lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8),Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQID NO: 10), Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQID NO: 12), Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQID NO: 14), Candida dubliniensis (SEQ ID NO: 15), Candida albicans (SEQID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis(SEQ ID NO: 18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger(SEQ ID NO: 20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomycespombe (SEQ ID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).

In one embodiment, the recombinant microorganism includes a mutation inat least one gene encoding for a 3-ketoacid reductase resulting in areduction of 3-ketoacid reductase activity of a polypeptide encoded bysaid gene. In another embodiment, the recombinant microorganism includesa partial deletion of gene encoding for a 3-ketoacid reductase resultingin a reduction of 3-ketoacid reductase activity of a polypeptide encodedby the gene. In another embodiment, the recombinant microorganismcomprises a complete deletion of a gene encoding for a 3-ketoacidreductase resulting in a reduction of 3-ketoacid reductase activity of apolypeptide encoded by the gene. In yet another embodiment, therecombinant microorganism includes a modification of the regulatoryregion associated with the gene encoding for a 3-ketoacid reductaseresulting in a reduction of expression of a polypeptide encoded by saidgene. In yet another embodiment, the recombinant microorganism comprisesa modification of the transcriptional regulator resulting in a reductionof transcription of a gene encoding for a 3-ketoacid reductase. In yetanother embodiment, the recombinant microorganism comprises mutations inall genes encoding for a 3-ketoacid reductase resulting in a reductionof activity of a polypeptide encoded by the gene(s). In one embodiment,the 3-ketoacid reductase activity or expression is reduced by at leastabout 50%. In another embodiment, the 3-ketoacid reductase activity orexpression is reduced by at least about 60%, by at least about 65%, byat least about 70%, by at least about 75%, by at least about 80%, by atleast about 85%, by at least about 90%, by at least about 95%, or by atleast about 99% as compared to a recombinant microorganism notcomprising a reduction of the 3-ketoacid reductase activity orexpression. In one embodiment, said 3-ketoacid reductase is encoded bythe S. cerevisiae TMA29 (YMR226C) gene or a homolog thereof.

In various embodiments described herein, the recombinant microorganismsof the invention may comprise a reduction or deletion of the activity orexpression of one or more endogenous proteins involved in catalyzing theconversion of an aldehyde to an acid by-product. In one embodiment, theactivity or expression of one or more endogenous proteins involved incatalyzing the conversion of an aldehyde to an acid by-product isreduced by at least about 50%. In another embodiment, the activity orexpression of one or more endogenous proteins involved in catalyzing theconversion of an aldehyde to an acid by-product is reduced by at leastabout 60%, by at least about 65%, by at least about 70%, by at leastabout 75%, by at least about 80%, by at least about 85%, by at leastabout 90%, by at least about 95%, or by at least about 99% as comparedto a recombinant microorganism not comprising a reduction or deletion ofthe activity or expression of one or more endogenous proteins involvedin catalyzing the conversion of an aldehyde to an acid by-product.

In various embodiments described herein, the endogenous protein involvedin catalyzing the conversion of an aldehyde to an acid by-product is analdehyde dehydrogenase (ALDH). In one embodiment, the aldehydedehydrogenase is encoded by a gene selected from the group consisting ofALD2, ALD3, ALD4, ALD5, ALD6, and HFD1, and homologs and variantsthereof. In an exemplary embodiment, the aldehyde dehydrogenase is theS. cerevisiae ALD6 (SEQ ID NO: 25) protein. In some embodiments, thealdehyde dehydrogenase is the S. cerevisiae ALD6 (SEQ ID NO: 25) proteinor a homolog or variant thereof. In one embodiment, the homolog isselected from the group consisting of Saccharomyces castelli (SEQ ID NO:26), Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO:28), Kluyveromyces lactis (SEQ ID NO: 29), Kluyveromyces thermotolerans(SEQ ID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomycescerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQID NO: 33), Saccharomyces cerevisiae EC1118 (SEQ ID NO: 34),Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomycescerevisiae AWR11631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a(SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38), Kluyveromycesmarxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40),and Schizosaccharomyces pombe (SEQ ID NO: 41).

In one embodiment, the recombinant microorganism includes a mutation inat least one gene encoding for an aldehyde dehydrogenase resulting in areduction of aldehyde dehydrogenase activity of a polypeptide encoded bysaid gene. In another embodiment, the recombinant microorganism includesa partial deletion of gene encoding for an aldehyde dehydrogenaseresulting in a reduction of aldehyde dehydrogenase activity of apolypeptide encoded by the gene. In another embodiment, the recombinantmicroorganism comprises a complete deletion of a gene encoding for analdehyde dehydrogenase resulting in a reduction of aldehydedehydrogenase activity of a polypeptide encoded by the gene. In yetanother embodiment, the recombinant microorganism includes amodification of the regulatory region associated with the gene encodingfor an aldehyde dehydrogenase resulting in a reduction of expression ofa polypeptide encoded by said gene. In yet another embodiment, therecombinant microorganism comprises a modification of thetranscriptional regulator resulting in a reduction of transcription of agene encoding for an aldehyde dehydrogenase. In yet another embodiment,the recombinant microorganism comprises mutations in all genes encodingfor an aldehyde dehydrogenase resulting in a reduction of activity of apolypeptide encoded by the gene(s). In one embodiment, the aldehydedehydrogenase activity or expression is reduced by at least about 50%.In another embodiment, the aldehyde dehydrogenase activity or expressionis reduced by at least about 60%, by at least about 65%, by at leastabout 70%, by at least about 75%, by at least about 80%, by at leastabout 85%, by at least about 90%, by at least about 95%, or by at leastabout 99% as compared to a recombinant microorganism not comprising areduction of the aldehyde dehydrogenase activity or expression. In oneembodiment, said aldehyde dehydrogenase is encoded by the S. cerevisiaeALD6 gene or a homolog thereof.

In various embodiments described herein, the recombinant microorganismmay comprise a biosynthetic pathway which uses a 3-keto acid as anintermediate. In one embodiment, the 3-keto acid intermediate isacetolactate. The biosynthetic pathway which uses acetolactate as anintermediate may be selected from a pathway for the biosynthesis ofisobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin,diacetyl, valine, leucine, pantothenic acid, isobutylene,3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. In anotherembodiment, the 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate.The biosynthetic pathway which uses 2-aceto-2-hydroxybutyrate as anintermediate may be selected from a pathway for the biosynthesis of2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol,and 5-methyl-1-heptanol.

In various embodiments described herein, the recombinant microorganismmay comprise a biosynthetic pathway which uses an aldehyde as anintermediate. The biosynthetic pathway which uses an aldehyde as anintermediate may be selected from a pathway for the biosynthesis ofisobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol,1-propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol,4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol. Invarious embodiments described herein, the aldehyde intermediate may beselected from isobutyraldehyde, 1-butanal, 2-methyl-1-butanal,3-methyl-1-butanal, 1-propanal, 1-pentanel, 1-hexanal,3-methyl-1-pentanal, 4-methyl-1-pentanal, 4-methyl-1-hexanal, and5-methyl-1-heptanal.

In various embodiments described herein, the recombinant microorganismmay comprise a biosynthetic pathway which uses a 3-keto acid and analdehyde as intermediates. In one embodiment, the 3-keto acidintermediate is acetolactate. The biosynthetic pathway which usesacetolactate and an aldehyde as intermediates may be selected from apathway for the biosynthesis of isobutanol, 1-butanol, and3-methyl-1-butanol. In another embodiment, the 3-keto acid intermediateis 2-aceto-2-hydroxybutyrate. The biosynthetic pathway which uses2-aceto-2-hydroxybutyrate and an aldehyde as intermediates may beselected from a pathway for the biosynthesis of 2-methyl-1-butanol,3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.

In one embodiment, the invention is directed to a recombinantmicroorganism for producing isobutanol, wherein said recombinantmicroorganism comprises an isobutanol producing metabolic pathway andwherein said microorganism is engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofacetolactate to DH2MB. In some embodiments, the enzyme catalyzing theconversion of acetolactate to DH2MB is a 3-ketoacid reductase (3-KAR).In a specific embodiment, the 3-ketoacid reductase is encoded by the S.cerevisiae TMA29 (YMR226C) gene or a homolog thereof.

In another embodiment, the invention is directed to a recombinantmicroorganism for producing isobutanol, wherein said recombinantmicroorganism comprises an isobutanol producing metabolic pathway andwherein said microorganism is engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofisobutyraldehyde to isobutyrate. In some embodiments, the enzymecatalyzing the conversion of isobutyraldehyde to isobutyrate is analdehyde dehydrogenase. In a specific embodiment, the aldehydedehydrogenase is encoded by the S. cerevisiae ALD6 gene or a homologthereof.

In yet another embodiment, the invention is directed to a recombinantmicroorganism for producing isobutanol, wherein said recombinantmicroorganism comprises an isobutanol producing metabolic pathway andwherein said microorganism is (i) engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofacetolactate to DH2MB and (ii) engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofisobutyraldehyde to isobutyrate. In some embodiments, the enzymecatalyzing the conversion of acetolactate to DH2MB is a 3-ketoacidreductase (3-KAR). In a specific embodiment, the 3-ketoacid reductase isencoded by the S. cerevisiae TMA29 (YMR226C) gene or a homolog thereof.In some embodiments, the enzyme catalyzing the conversion ofisobutyraldehyde to isobutyrate is an aldehyde dehydrogenase. In aspecific embodiment, the aldehyde dehydrogenase is encoded by the S.cerevisiae ALD6 gene or a homolog thereof.

In one embodiment, the isobutanol producing metabolic pathway comprisesat least one exogenous gene that catalyzes a step in the conversion ofpyruvate to isobutanol. In another embodiment, the isobutanol producingmetabolic pathway comprises at least two exogenous genes that catalyzesteps in the conversion of pyruvate to isobutanol. In yet anotherembodiment, the isobutanol producing metabolic pathway comprises atleast three exogenous genes that catalyze steps in the conversion ofpyruvate to isobutanol. In yet another embodiment, the isobutanolproducing metabolic pathway comprises at least four exogenous genes thatcatalyze steps in the conversion of pyruvate to isobutanol. In yetanother embodiment, the isobutanol producing metabolic pathway comprisesat five exogenous genes that catalyze steps in the conversion ofpyruvate to isobutanol.

In one embodiment, one or more of the isobutanol pathway genes encodesan enzyme that is localized to the cytosol. In one embodiment, therecombinant microorganisms comprise an isobutanol producing metabolicpathway with at least one isobutanol pathway enzyme localized in thecytosol. In another embodiment, the recombinant microorganisms comprisean isobutanol producing metabolic pathway with at least two isobutanolpathway enzymes localized in the cytosol. In yet another embodiment, therecombinant microorganisms comprise an isobutanol producing metabolicpathway with at least three isobutanol pathway enzymes localized in thecytosol. In yet another embodiment, the recombinant microorganismscomprise an isobutanol producing metabolic pathway with at least fourisobutanol pathway enzymes localized in the cytosol. In an exemplaryembodiment, the recombinant microorganisms comprise an isobutanolproducing metabolic pathway with five isobutanol pathway enzymeslocalized in the cytosol.

In various embodiments described herein, the isobutanol pathway genesencodes enzyme(s) selected from the group consisting of acetolactatesynthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxyaciddehydratase (DHAD), 2-keto-acid decarboxylase (KIVD), and alcoholdehydrogenase (ADH).

In another aspect, the recombinant microorganism may be engineered toreduce the conversion of isobutanol to isobutyraldehyde by reducingand/or eliminating the expression of one or more alcohol dehydrogenases.In a specific embodiment, the alcohol dehydrogenase is encoded by a geneselected from the group consisting of ADH1, ADH2, ADH3, ADH4, ADH5,ADH6, and ADH7, and homologs and variants thereof.

In another aspect, the present invention relates to modified alcoholdehydrogenase (ADH) enzymes that exhibit an enhanced ability to convertisobutyraldehyde to isobutanol. In general, cells expressing theseimproved ADH enzymes will produce increased levels of isobutanol duringfermentation reactions. While the modified ADH enzymes of the presentinvention have utility in isobutanol-producing fermentation reactions,it will be understood by those skilled in the art equipped with thisdisclosure that the modified ADH enzymes also have usefulness infermentation reactions producing other alcohols such as 1-propanol,2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-methyl-1-butanol, and3-methyl-1-butanol.

In certain aspects, the invention is directed to alcohol dehydrogenases(ADHs), which have been modified to enhance the enzyme's ability toconvert isobutyraldehyde to isobutanol. Examples of such ADHs includeenzymes having one or more mutations at positions corresponding to aminoacids selected from: (a) tyrosine 50 of the L. lactis AdhA (SEQ ID NO:185); (b) glutamine 77 of the L. lactis AdhA (SEQ ID NO: 185); (c)valine 108 of the L. lactis AdhA (SEQ ID NO: 185); (d) tyrosine 113 ofthe L. lactis AdhA (SEQ ID NO: 185); (e) isoleucine 212 of the L. lactisAdhA (SEQ ID NO: 185); and (f) leucine 264 of the L. lactis AdhA (SEQ IDNO: 185), wherein AdhA (SEQ ID NO: 185) is encoded by the L. lactisalcohol dehydrogenase (ADH) gene adhA (SEQ ID NO: 184) or acodon-optimized version thereof (SEQ ID NO: 206).

In one embodiment, the modified ADH enzyme contains a mutation at theamino acid corresponding to position 50 of the L. lactis AdhA (SEQ IDNO: 185). In another embodiment, the modified ADH enzyme contains amutation at the amino acid corresponding to position 77 of the L. lactisAdhA (SEQ ID NO: 185). In yet another embodiment, the modified ADHenzyme contains a mutation at the amino acid corresponding to position108 of the L. lactis AdhA (SEQ ID NO: 185). In yet another embodiment,the modified ADH enzyme contains a mutation at the amino acidcorresponding to position 113 of the L. lactis AdhA (SEQ ID NO: 185). Inyet another embodiment, the modified ADH enzyme contains a mutation atthe amino acid corresponding to position 212 of the L. lactis AdhA (SEQID NO: 185). In yet another embodiment, the modified ADH enzyme containsa mutation at the amino acid corresponding to position 264 of the L.lactis AdhA (SEQ ID NO: 185).

In one embodiment, the ADH enzyme contains two or more mutations at theamino acids corresponding to the positions described above. In anotherembodiment, the ADH enzyme contains three or more mutations at the aminoacids corresponding to the positions described above. In yet anotherembodiment, the ADH enzyme contains four or more mutations at the aminoacids corresponding to the positions described above. In yet anotherembodiment, the ADH enzyme contains five or more mutations at the aminoacids corresponding to the positions described above. In yet anotherembodiment, the ADH enzyme contains six mutations at the amino acidscorresponding to the positions described above.

In one specific embodiment, the invention is directed to ADH enzymeswherein the tyrosine at position 50 is replaced with a phenylalanine ortryptophan residue. In another specific embodiment, the invention isdirected to ADH enzymes wherein the glutamine at position 77 is replacedwith an arginine or serine residue. In another specific embodiment, theinvention is directed to ADH enzymes wherein the valine at position 108is replaced with a serine or alanine residue. In another specificembodiment, the invention is directed to ADH enzymes wherein thetyrosine at position 113 is replaced with a phenylalanine or glycineresidue. In another specific embodiment, the invention is directed toADH enzymes wherein the isoleucine at position 212 is replaced with athreonine or valine residue. In yet another specific embodiment, theinvention is directed to ADH enzymes wherein the leucine at position 264is replaced with a valine residue. In one embodiment, the ADH enzymecontains two or more mutations at the amino acids corresponding to thepositions described in these specific embodiments. In anotherembodiment, the ADH enzyme contains three or more mutations at the aminoacids corresponding to the positions described in these specificembodiments. In yet another embodiment, the ADH enzyme contains four ormore mutations at the amino acids corresponding to the positionsdescribed in these specific embodiments. In yet another embodiment, theADH enzyme contains five or more mutations at the amino acidscorresponding to the positions described in these specific embodiments.In yet another embodiment, the ADH enzyme contains six mutations at theamino acids corresponding to the positions described in these specificembodiments.

In certain exemplary embodiments, the ADH enzyme comprises a sequenceselected SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195,SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ IDNO: 205, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214,SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ IDNO: 224, and homologs or variants thereof comprising correspondingmutations as compared to the wild-type or parental enzyme.

As alluded to in the preceding paragraph, further included within thescope of the invention are ADH enzymes, other than the L. lactis AdhA(SEQ ID NO: 185), which contain alterations corresponding to those setout above. Such ADH enzymes may include, but are not limited to, the ADHenzymes listed in Table 97.

In some embodiments, the ADH enzymes to be modified are NADH-dependentADH enzymes. Examples of such NADH-dependent ADH enzymes are describedin commonly owned and co-pending U.S. Patent Publication No.2010/0143997, which is herein incorporated by reference in its entiretyfor all purposes. In some embodiments, genes originally encodingNADPH-utilizing ADH enzymes are modified to switch the co-factorpreference of the enzyme to NADH.

As described herein, the modified ADHs will generally exhibit anenhanced ability to convert isobutyraldehyde to isobutanol as comparedto the wild-type or parental ADH. Preferably, the catalytic efficiency(k_(cat)/K_(M)) of the modified ADH enzyme is enhanced by at least about5% as compared to the wild-type or parental ADH. More preferably, thecatalytic efficiency of the modified ADH enzyme is enhanced by at leastabout 15% as compared to the wild-type or parental ADH. More preferably,the catalytic efficiency of the modified ADH enzyme is enhanced by atleast about 25% as compared to the wild-type or parental ADH. Morepreferably, the catalytic efficiency of the modified ADH enzyme isenhanced by at least about 50% as compared to the wild-type or parentalADH. More preferably, the catalytic efficiency of the modified ADHenzyme is enhanced by at least about 75% as compared to the wild-type orparental ADH. More preferably, the catalytic efficiency of the modifiedADH enzyme is enhanced by at least about 100% as compared to thewild-type or parental ADH. More preferably, the catalytic efficiency ofthe modified ADH enzyme is enhanced by at least about 200% as comparedto the wild-type or parental ADH. More preferably, the catalyticefficiency of the modified ADH enzyme is enhanced by at least about 500%as compared to the wild-type or parental ADH. More preferably, thecatalytic efficiency of the modified ADH enzyme is enhanced by at leastabout 1000% as compared to the wild-type or parental ADH. Morepreferably, the catalytic efficiency of the modified ADH enzyme isenhanced by at least about 2000% as compared to the wild-type orparental ADH. More preferably, the catalytic efficiency of the modifiedADH enzyme is enhanced by at least about 3000% as compared to thewild-type or parental ADH. Most preferably, the catalytic efficiency ofthe modified ADH enzyme is enhanced by at least about 3500% as comparedto the wild-type or parental ADH.

In additional aspects, the invention is directed to modified ADH enzymesthat have been codon optimized for expression in certain desirable hostorganisms, such as yeast and E. coli. In other aspects, the presentinvention is directed to recombinant host cells comprising a modifiedADH enzyme of the invention. According to this aspect, the presentinvention is also directed to methods of using the modified ADH enzymesin any fermentation process, where the conversion of isobutyraldehyde toisobutanol is desired. In one embodiment according to this aspect, themodified ADH enzymes may be suitable for enhancing a host cell's abilityto produce isobutanol. In another embodiment according to this aspect,the modified ADH enzymes may be suitable for enhancing a host cell'sability to produce 1-propanol, 2-propanol, 1-butanol, 2-butanol,1-pentanol, 2-methyl-1-butanol, and 3-methyl-1-butanol.

In various embodiments described herein, the recombinant microorganismscomprising a modified ADH may be further engineered to express anisobutanol producing metabolic pathway. In one embodiment, therecombinant microorganism may be engineered to express an isobutanolproducing metabolic pathway comprising at least one exogenous gene. Inone embodiment, the recombinant microorganism may be engineered toexpress an isobutanol producing metabolic pathway comprising at leasttwo exogenous genes. In another embodiment, the recombinantmicroorganism may be engineered to express an isobutanol producingmetabolic pathway comprising at least three exogenous genes. In anotherembodiment, the recombinant microorganism may be engineered to expressan isobutanol producing metabolic pathway comprising at least fourexogenous genes. In another embodiment, the recombinant microorganismmay be engineered to express an isobutanol producing metabolic pathwaycomprising five exogenous genes. Thus, the present invention furtherprovides recombinant microorganisms that comprise an isobutanolproducing metabolic pathway and methods of using said recombinantmicroorganisms to produce isobutanol.

In various embodiments described herein, the isobutanol pathwayenzyme(s) is/are selected from acetolactate synthase (ALS), ketol-acidreductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2-keto-aciddecarboxylase (KIVD), and alcohol dehydrogenase (ADH).

In various embodiments described herein, the isobutanol pathway enzymesmay be derived from a prokaryotic organism. In alternative embodimentsdescribed herein, the isobutanol pathway enzymes may be derived from aeukaryotic organism. An exemplary metabolic pathway that convertspyruvate to isobutanol may be comprised of a acetohydroxy acid synthase(ALS) enzyme encoded by, for example, alsS from B. subtilis, aketol-acid reductoisomerase (KARI) encoded by, for example ilvC from E.coli, a dihydroxy-acid dehydratase (DHAD), encoded by, for example, ilvDfrom L. lactis, a 2-keto-acid decarboxylase (KIVD) encoded by, forexample kivD from L. lactis, and an alcohol dehydrogenase (ADH) (e.g. amodified ADH described herein), encoded by, for example, adhA from L.lactis with one or more mutations at positions Y50, Q77, V108, Y113,I212, and L264 as described herein.

In various embodiments described herein, the recombinant microorganismsmay be microorganisms of the Saccharomyces clade, Saccharomyces sensustricto microorganisms, Crabtree-negative yeast microorganisms,Crabtree-positive yeast microorganisms, post-WGD (whole genomeduplication) yeast microorganisms, pre-WGD (whole genome duplication)yeast microorganisms, and non-fermenting yeast microorganisms.

In some embodiments, the recombinant microorganisms may be yeastrecombinant microorganisms of the Saccharomyces clade.

In some embodiments, the recombinant microorganisms may be Saccharomycessensu stricto microorganisms. In one embodiment, the Saccharomyces sensustricto is selected from the group consisting of S. cerevisiae, S.kudriavzevii, S. mikatae, S. bayanus, S. uvarum. S. carocanis andhybrids thereof.

In some embodiments, the recombinant microorganisms may beCrabtree-negative recombinant yeast microorganisms. In one embodiment,the Crabtree-negative yeast microorganism is classified into a generaselected from the group consisting of Saccharomyces, Kluyveromyces,Pichia, Issatchenkia, Hansenula, or Candida. In additional embodiments,the Crabtree-negative yeast microorganism is selected from Saccharomyceskluyveri, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia anomala,Pichia stipitis, Hansenula anomala, Candida utilis and Kluyveromyceswaltii.

In some embodiments, the recombinant microorganisms may beCrabtree-positive recombinant yeast microorganisms. In one embodiment,the Crabtree-positive yeast microorganism is classified into a generaselected from the group consisting of Saccharomyces, Kluyveromyces,Zygosaccharomyces, Debaryomyces, Candida, Pichia andSchizosaccharomyces. In additional embodiments, the Crabtree-positiveyeast microorganism is selected from the group consisting ofSaccharomyces cerevisiae, Saccharomyces uvarum, Saccharomyces bayanus,Saccharomyces paradoxus, Saccharomyces castelli, Kluyveromycesthermotolerans, Candida glabrata, Z. bailli, Z. rouxii, Debaryomyceshansenii, Pichia pastorius, Schizosaccharomyces pombe, and Saccharomycesuvarum.

In some embodiments, the recombinant microorganisms may be post-WGD(whole genome duplication) yeast recombinant microorganisms. In oneembodiment, the post-WGD yeast recombinant microorganism is classifiedinto a genera selected from the group consisting of Saccharomyces orCandida. In additional embodiments, the post-WGD yeast is selected fromthe group consisting of Saccharomyces cerevisiae, Saccharomyces uvarum,Saccharomyces bayanus, Saccharomyces paradoxus, Saccharomyces castelli,and Candida glabrata.

In some embodiments, the recombinant microorganisms may be pre-WGD(whole genome duplication) yeast recombinant microorganisms. In oneembodiment, the pre-WGD yeast recombinant microorganism is classifiedinto a genera selected from the group consisting of Saccharomyces,Kluyveromyces, Candida, Pichia, Issatchenkia, Debaryomyces, Hansenula,Pachysolen, Yarrowia and Schizosaccharomyces. In additional embodiments,the pre-WGD yeast is selected from the group consisting of Saccharomyceskluyveri, Kluyveromyces thermotolerans, Kluyveromyces marxianus,Kluyveromyces waltii, Kluyveromyces lactis, Candida tropicalis, Pichiapastoris, Pichia anomala, Pichia stipitis, Issatchenkia orientalis,Issatchenkia occidentalis, Debaryomyces hansenii, Hansenula anomala,Pachysolen tannophilis, Yarrowia lipolytica, and Schizosaccharomycespombe.

In some embodiments, the recombinant microorganisms may bemicroorganisms that are non-fermenting yeast microorganisms, including,but not limited to those, classified into a genera selected from thegroup consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida. In aspecific embodiment, the non-fermenting yeast is C. xestobii.

In another aspect, the present invention provides methods of producingbeneficial metabolites including fuels, chemicals, and amino acids usinga recombinant microorganism as described herein. In one embodiment, themethod includes cultivating the recombinant microorganism in a culturemedium containing a feedstock providing the carbon source until arecoverable quantity of the metabolite is produced and optionally,recovering the metabolite. In one embodiment, the microorganism producesthe metabolite from a carbon source at a yield of at least about 5percent theoretical. In another embodiment, the microorganism producesthe metabolite at a yield of at least about 10 percent, at least about15 percent, about least about 20 percent, at least about 25 percent, atleast about 30 percent, at least about 35 percent, at least about 40percent, at least about 45 percent, at least about 50 percent, at leastabout 55 percent, at least about 60 percent, at least about 65 percent,at least about 70 percent, at least about 75 percent, at least about 80percent, at least about 85 percent, at least about 90 percent, at leastabout 95 percent, or at least about 97.5 percent theoretical. In oneembodiment, the metabolite may be derived from a biosynthetic pathwaywhich uses a 3-keto acid as an intermediate. In one embodiment, the3-keto acid intermediate is acetolactate. Accordingly, the metabolitemay be derived from a biosynthetic pathway which uses acetolactate as anintermediate, including, but not limited to, isobutanol, 2-butanol,1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine,leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol,4-methyl-1-pentanol, and coenzyme A. In another embodiment, the 3-ketoacid intermediate is 2-aceto-2-hydroxybutyrate. Accordingly, themetabolite may be derived from a biosynthetic pathway which uses2-aceto-2-hydroxybutyrate as an intermediate, including, but not limitedto, 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol,4-methyl-1-hexanol, and 5-methyl-1-heptanol. In another embodiment, themetabolite may be derived from a biosynthetic pathway which uses analdehyde as an intermediate, including, but not limited to, isobutanol,1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol,1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol,4-methyl-1-hexanol, and 5-methyl-1-heptanol. In yet another embodiment,the metabolite may be derived from a biosynthetic pathway which usesacetolactate and an aldehyde as intermediates, including, but notlimited to, isobutanol, 1-butanol, and 3-methyl-1-butanol biosyntheticpathways. In yet another embodiment, the metabolite may be derived froma biosynthetic pathway which uses 2-aceto-2-hydroxybutyrate and analdehyde as intermediates, including, but not limited to,2-methyl-1-butanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and5-methyl-1-heptanol biosynthetic pathways.

In one embodiment, the recombinant microorganism is grown under aerobicconditions. In another embodiment, the recombinant microorganism isgrown under microaerobic conditions. In yet another embodiment, therecombinant microorganism is grown under anaerobic conditions.

BRIEF DESCRIPTION OF DRAWINGS

Illustrative embodiments of the invention are illustrated in thedrawings, in which:

FIG. 1 illustrates an exemplary embodiment of an isobutanol pathway.

FIG. 2 illustrates exemplary reactions capable of being catalyzed by3-ketoacid reductases.

FIG. 3 illustrates a non-limiting list of exemplary 3-ketoacidreductases and their corresponding enzyme classification numbers.

FIG. 4 illustrates exemplary reactions capable of being catalyzed byaldehyde dehydrogenases.

FIG. 5 illustrates a strategy for reducing the production of DH2MB andisobutyrate in isobutanol-producing recombinant microorganisms.

FIG. 6 illustrates a strategy for reducing the production of DH2MB and3-methyl-1-butyrate in 3-methyl-1-butanol-producing recombinantmicroorganisms.

FIG. 7 illustrates a strategy for reducing the production of2-ethyl-2,3-dihydroxybutyrate and 2-methyl-1-butyrate in2-methyl-1-butanol producing recombinant microorganisms.

FIG. 8 illustrates a stacked overlay of LC4 chromatograms showing asample containing DH2MB and acetate (top) and a sample containingacetate and DHIV (bottom). Elution order: DH2MB followed by acetate(top); lactate, acetate, DHIV, isobutyrate, pyruvate (bottom).

FIG. 9 illustrates a chromatogram for sample fraction collected atretention time corresponding to DHIV collected on LC1 and analyzed byLC4 on an AS-11 Column with Conductivity Detection.

FIG. 10 illustrates a 1H-COSY spectrum of the peak isolated from LC1.The spectrum indicates that DH2MB methyl protons (doublet) at 0.95 ppmare coupled to methine proton (quartet) at 3.7 ppm.

FIG. 11 illustrates a 1H-NMR spectrum of the peak isolated from LC1. Thespectrum indicates the presence of DH2MB: a singlet of methyl protons(a) at 1.2 ppm with integral value 3, a doublet of methyl protons (b) at0.95 ppm with integral value 3 and a quartet of methine proton (c) at3.7 ppm with integral value of 1.84. Integral value of methine proton(c) is greater than 1 due to overlap with glucose resonance in the sameregion.

FIG. 12 illustrates a LC-MS analysis of the peak isolated from LC1.Several molecular ions were identified in the sample as indicated at thetop portion of the figure. Further fragmentation (MS2) of 134 molecularion indicated that isolated LC1 fraction contains hydroxyl carboxylicacid by characteristic loss of CO₂ (*) and H₂O+CO₂ (**).

FIG. 13 illustrates the diastereomeric and enantiomeric structures of2,3-dihydroxy-2-methylbutanoic acid (2R,3S)-1a, (2S,3R)-1b, (2R,3R)-2a,(2S,3S)-2b.

FIG. 14 illustrates the 1H spectrum of crystallized DH2MB in D₂O. 1H NMR(TSP) 1.1 (d, 6.5 Hz, 3H), 1.3 (s, 3H), 3.9 (q, 6.5 Hz, 3H)

FIG. 15 illustrates the 13C spectrum of crystallized DH2MB in D₂O. Thespectrum indicates five different carbon resonances one of them beingcharacteristic carboxylic acid resonance at 181 ppm.

FIG. 16 illustrates the fermentation profile of isobutanol andby-products from a single fermentation with GEVO3160. Productionaeration was reduced from an OTR of 0.8 mM/h to 0.3 mM/h at 93 h postinoculation. Open diamond=iBuOH, square=unknown quantified as DH2MB,asterisk=cell dry weight (cdw), and closed triangle=total byproducts.

FIG. 17 illustrates a structural alignment of the L. lactis AdhA aminoacid sequence with the structure of G. stearothermophilus (Pymol).Active site mutations are shown (Y50F and L264V). Mutations in theco-factor binding domain are also shown (I212T and N219Y).

FIG. 18 illustrates biosynthetic pathways utilizing acetolactate as anintermediate. Biosynthetic pathways for the production of 1-butanol,isobutanol, 3-methyl-1-butanol, and 4-methyl-1-pentanol use bothacetolactate and an aldehyde as an intermediate.

FIG. 19 illustrates biosynthetic pathways utilizing2-aceto-2-hydroxybutyrate as an intermediate. Biosynthetic pathways forthe production of 2-methyl-1-butanol, 3-methyl-1-pentanol,4-methyl-1-hexanol, and 5-methyl-1-heptanol use both2-aceto-2-hydroxybutyrate and an aldehyde as an intermediate.

FIG. 20 illustrates additional biosynthetic pathways utilizing analdehyde as an intermediate.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a polynucleotide” includes aplurality of such polynucleotides and reference to “the microorganism”includes reference to one or more microorganisms, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

Any publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

The term “microorganism” includes prokaryotic and eukaryotic microbialspecies from the Domains Archaea, Bacteria and Eucarya, the latterincluding yeast and filamentous fungi, protozoa, algae, or higherProtista. The terms “microbial cells” and “microbes” are usedinterchangeably with the term microorganism.

The term “genus” is defined as a taxonomic group of related speciesaccording to the Taxonomic Outline of Bacteria and Archaea (Garrity, G.M., Lilburn, T. G., Cole, J. R., Harrison, S. H., Euzeby, J., andTindall, B. J. (2007) The Taxonomic Outline of Bacteria and Archaea.TOBA Release 7.7, March 2007. Michigan State University Board ofTrustees. [http://www.taxonomicoutline.org/]).

The term “species” is defined as a collection of closely relatedorganisms with greater than 97% 16S ribosomal RNA sequence homology andgreater than 70% genomic hybridization and sufficiently different fromall other organisms so as to be recognized as a distinct unit.

The terms “recombinant microorganism,” “modified microorganism,” and“recombinant host cell” are used interchangeably herein and refer tomicroorganisms that have been genetically modified to express oroverexpress endogenous polynucleotides, to express heterologouspolynucleotides, such as those included in a vector, in an integrationconstruct, or which have an alteration in expression of an endogenousgene. By “alteration” it is meant that the expression of the gene, orlevel of a RNA molecule or equivalent RNA molecules encoding one or morepolypeptides or polypeptide subunits, or activity of one or morepolypeptides or polypeptide subunits is up regulated or down regulated,such that expression, level, or activity is greater than or less thanthat observed in the absence of the alteration. For example, the term“alter” can mean “inhibit,” but the use of the word “alter” is notlimited to this definition.

The term “expression” with respect to a gene sequence refers totranscription of the gene and, as appropriate, translation of theresulting mRNA transcript to a protein. Thus, as will be clear from thecontext, expression of a protein results from transcription andtranslation of the open reading frame sequence. The level of expressionof a desired product in a host cell may be determined on the basis ofeither the amount of corresponding mRNA that is present in the cell, orthe amount of the desired product encoded by the selected sequence. Forexample, mRNA transcribed from a selected sequence can be quantitated byqRT-PCR or by Northern hybridization (see Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1989)). Protein encoded by a selected sequence can be quantitated byvarious methods, e.g., by ELISA, by assaying for the biological activityof the protein, or by employing assays that are independent of suchactivity, such as western blotting or radioimmunoassay, using antibodiesthat recognize and bind the protein. See Sambrook et al., 1989, supra.The polynucleotide generally encodes a target enzyme involved in ametabolic pathway for producing a desired metabolite. It is understoodthat the terms “recombinant microorganism” and “recombinant host cell”refer not only to the particular recombinant microorganism but to theprogeny or potential progeny of such a microorganism. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The term “overexpression” refers to an elevated level (e.g., aberrantlevel) of mRNAs encoding for a protein(s) (e.g., an TMA29 protein orhomolog thereof), and/or to elevated levels of protein(s) (e.g., TMA29)in cells as compared to similar corresponding unmodified cellsexpressing basal levels of mRNAs (e.g., those encoding Aft proteins) orhaving basal levels of proteins. In particular embodiments, TMA29, orhomologs thereof, may be overexpressed by at least 2-fold, 3-fold,4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold or more inmicroorganisms engineered to exhibit increased TMA29 mRNA, protein,and/or activity.

As used herein and as would be understood by one of ordinary skill inthe art, “reduced activity and/or expression” of a protein such as anenzyme can mean either a reduced specific catalytic activity of theprotein (e.g. reduced activity) and/or decreased concentrations of theprotein in the cell (e.g. reduced expression), while “deleted activityand/or expression” or “eliminated activity and/or expression” of aprotein such as an enzyme can mean either no or negligible specificcatalytic activity of the enzyme (e.g. deleted activity) and/or no ornegligible concentrations of the enzyme in the cell (e.g. deletedexpression).

The term “wild-type microorganism” describes a cell that occurs innature, i.e. a cell that has not been genetically modified. A wild-typemicroorganism can be genetically modified to express or overexpress afirst target enzyme. This microorganism can act as a parentalmicroorganism in the generation of a microorganism modified to expressor overexpress a second target enzyme. In turn, the microorganismmodified to express or overexpress a first and a second target enzymecan be modified to express or overexpress a third target enzyme.

Accordingly, a “parental microorganism” functions as a reference cellfor successive genetic modification events. Each modification event canbe accomplished by introducing a nucleic acid molecule in to thereference cell. The introduction facilitates the expression oroverexpression of a target enzyme. It is understood that the term“facilitates” encompasses the activation of endogenous polynucleotidesencoding a target enzyme through genetic modification of e.g., apromoter sequence in a parental microorganism. It is further understoodthat the term “facilitates” encompasses the introduction of heterologouspolynucleotides encoding a target enzyme in to a parental microorganism

The term “engineer” refers to any manipulation of a microorganism thatresults in a detectable change in the microorganism, wherein themanipulation includes but is not limited to inserting a polynucleotideand/or polypeptide heterologous to the microorganism and mutating apolynucleotide and/or polypeptide native to the microorganism.

The term “mutation” as used herein indicates any modification of anucleic acid and/or polypeptide which results in an altered nucleic acidor polypeptide. Mutations include, for example, point mutations,deletions, or insertions of single or multiple residues in apolynucleotide, which includes alterations arising within aprotein-encoding region of a gene as well as alterations in regionsoutside of a protein-encoding sequence, such as, but not limited to,regulatory or promoter sequences. A genetic alteration may be a mutationof any type. For instance, the mutation may constitute a point mutation,a frame-shift mutation, a nonsense mutation, an insertion, or a deletionof part or all of a gene. In addition, in some embodiments of themodified microorganism, a portion of the microorganism genome has beenreplaced with a heterologous polynucleotide. In some embodiments, themutations are naturally-occurring. In other embodiments, the mutationsare identified and/or enriched through artificial selection pressure. Instill other embodiments, the mutations in the microorganism genome arethe result of genetic engineering.

The term “biosynthetic pathway”, also referred to as “metabolicpathway”, refers to a set of anabolic or catabolic biochemical reactionsfor converting one chemical species into another. Gene products belongto the same “metabolic pathway” if they, in parallel or in series, acton the same substrate, produce the same product, or act on or produce ametabolic intermediate (i.e., metabolite) between the same substrate andmetabolite end product.

As used herein, the term “isobutanol producing metabolic pathway” refersto an enzyme pathway which produces isobutanol from pyruvate.

The term “heterologous” as used herein with reference to molecules andin particular enzymes and polynucleotides, indicates molecules that areexpressed in an organism other than the organism from which theyoriginated or are found in nature, independently of the level ofexpression that can be lower, equal or higher than the level ofexpression of the molecule in the native microorganism.

On the other hand, the term “native” or “endogenous” as used herein withreference to molecules, and in particular enzymes and polynucleotides,indicates molecules that are expressed in the organism in which theyoriginated or are found in nature, independently of the level ofexpression that can be lower equal or higher than the level ofexpression of the molecule in the native microorganism. It is understoodthat expression of native enzymes or polynucleotides may be modified inrecombinant microorganisms.

The term “feedstock” is defined as a raw material or mixture of rawmaterials supplied to a microorganism or fermentation process from whichother products can be made. For example, a carbon source, such asbiomass or the carbon compounds derived from biomass are a feedstock fora microorganism that produces a biofuel in a fermentation process.However, a feedstock may contain nutrients other than a carbon source.

The term “substrate” or “suitable substrate” refers to any substance orcompound that is converted or meant to be converted into anothercompound by the action of an enzyme. The term includes not only a singlecompound, but also combinations of compounds, such as solutions,mixtures and other materials which contain at least one substrate, orderivatives thereof. Further, the term “substrate” encompasses not onlycompounds that provide a carbon source suitable for use as a startingmaterial, such as any biomass derived sugar, but also intermediate andend product metabolites used in a pathway associated with a recombinantmicroorganism as described herein.

The term “C2-compound” as used as a carbon source for engineered yeastmicroorganisms with mutations in all pyruvate decarboxylase (PDC) genesresulting in a reduction of pyruvate decarboxylase activity of saidgenes refers to organic compounds comprised of two carbon atoms,including but not limited to ethanol and acetate.

The term “fermentation” or “fermentation process” is defined as aprocess in which a microorganism is cultivated in a culture mediumcontaining raw materials, such as feedstock and nutrients, wherein themicroorganism converts raw materials, such as a feedstock, intoproducts.

The term “volumetric productivity” or “production rate” is defined asthe amount of product formed per volume of medium per unit of time.Volumetric productivity is reported in gram per liter per hour (g/L/h).

The term “specific productivity” or “specific production rate” isdefined as the amount of product formed per volume of medium per unit oftime per amount of cells. Specific productivity is reported in gram ormilligram per liter per hour per OD (g/L/h/OD).

The term “yield” is defined as the amount of product obtained per unitweight of raw material and may be expressed as g product per g substrate(g/g). Yield may be expressed as a percentage of the theoretical yield.“Theoretical yield” is defined as the maximum amount of product that canbe generated per a given amount of substrate as dictated by thestoichiometry of the metabolic pathway used to make the product. Forexample, the theoretical yield for one typical conversion of glucose toisobutanol is 0.41 g/g. As such, a yield of isobutanol from glucose of0.39 g/g would be expressed as 95% of theoretical or 95% theoreticalyield.

The term “titer” is defined as the strength of a solution or theconcentration of a substance in solution. For example, the titer of abiofuel in a fermentation broth is described as g of biofuel in solutionper liter of fermentation broth (g/L).

“Aerobic conditions” are defined as conditions under which the oxygenconcentration in the fermentation medium is sufficiently high for anaerobic or facultative anaerobic microorganism to use as a terminalelectron acceptor.

In contrast, “anaerobic conditions” are defined as conditions underwhich the oxygen concentration in the fermentation medium is too low forthe microorganism to use as a terminal electron acceptor. Anaerobicconditions may be achieved by sparging a fermentation medium with aninert gas such as nitrogen until oxygen is no longer available to themicroorganism as a terminal electron acceptor. Alternatively, anaerobicconditions may be achieved by the microorganism consuming the availableoxygen of the fermentation until oxygen is unavailable to themicroorganism as a terminal electron acceptor. Methods for theproduction of isobutanol under anaerobic conditions are described incommonly owned and co-pending publication, US 2010/0143997, thedisclosures of which are herein incorporated by reference in itsentirety for all purposes.

“Aerobic metabolism” refers to a biochemical process in which oxygen isused as a terminal electron acceptor to make energy, typically in theform of ATP, from carbohydrates. Aerobic metabolism occurs e.g. viaglycolysis and the TCA cycle, wherein a single glucose molecule ismetabolized completely into carbon dioxide in the presence of oxygen.

In contrast, “anaerobic metabolism” refers to a biochemical process inwhich oxygen is not the final acceptor of electrons contained in NADH.Anaerobic metabolism can be divided into anaerobic respiration, in whichcompounds other than oxygen serve as the terminal electron acceptor, andsubstrate level phosphorylation, in which the electrons from NADH areutilized to generate a reduced product via a “fermentative pathway.”

In “fermentative pathways”, NAD(P)H donates its electrons to a moleculeproduced by the same metabolic pathway that produced the electronscarried in NAD(P)H. For example, in one of the fermentative pathways ofcertain yeast strains, NAD(P)H generated through glycolysis transfersits electrons to pyruvate, yielding ethanol. Fermentative pathways areusually active under anaerobic conditions but may also occur underaerobic conditions, under conditions where NADH is not fully oxidizedvia the respiratory chain. For example, above certain glucoseconcentrations, Crabtree positive yeasts produce large amounts ofethanol under aerobic conditions.

The term “byproduct” or “by-product” means an undesired product relatedto the production of an amino acid, amino acid precursor, chemical,chemical precursor, biofuel, or biofuel precursor.

The term “substantially free” when used in reference to the presence orabsence of enzymatic activities (3-KAR, ALDH, PDC, GPD, etc.) in carbonpathways that compete with the desired metabolic pathway (e.g., anisobutanol-producing metabolic pathway) means the level of the enzyme issubstantially less than that of the same enzyme in the wild-type host,wherein less than about 50% of the wild-type level is preferred and lessthan about 30% is more preferred. The activity may be less than about20%, less than about 10%, less than about 5%, or less than about 1% ofwild-type activity. Microorganisms which are “substantially free” of aparticular enzymatic activity (3-KAR, ALDH, PDC, GPD, etc.) may becreated through recombinant means or identified in nature.

The term “non-fermenting yeast” is a yeast species that fails todemonstrate an anaerobic metabolism in which the electrons from NADH areutilized to generate a reduced product via a fermentative pathway suchas the production of ethanol and CO₂ from glucose. Non-fermentativeyeast can be identified by the “Durham Tube Test” (J. A. Barnett, R. W.Payne, and D. Yarrow. 2000. Yeasts Characteristics and Identification.3^(rd) edition. p. 28-29. Cambridge University Press, Cambridge, UK) orby monitoring the production of fermentation productions such as ethanoland CO₂.

The term “polynucleotide” is used herein interchangeably with the term“nucleic acid” and refers to an organic polymer composed of two or moremonomers including nucleotides, nucleosides or analogs thereof,including but not limited to single stranded or double stranded, senseor antisense deoxyribonucleic acid (DNA) of any length and, whereappropriate, single stranded or double stranded, sense or antisenseribonucleic acid (RNA) of any length, including siRNA. The term“nucleotide” refers to any of several compounds that consist of a riboseor deoxyribose sugar joined to a purine or a pyrimidine base and to aphosphate group, and that are the basic structural units of nucleicacids. The term “nucleoside” refers to a compound (as guanosine oradenosine) that consists of a purine or pyrimidine base combined withdeoxyribose or ribose and is found especially in nucleic acids. The term“nucleotide analog” or “nucleoside analog” refers, respectively, to anucleotide or nucleoside in which one or more individual atoms have beenreplaced with a different atom or with a different functional group.Accordingly, the term polynucleotide includes nucleic acids of anylength, DNA, RNA, analogs and fragments thereof. A polynucleotide ofthree or more nucleotides is also called nucleotidic oligomer oroligonucleotide.

It is understood that the polynucleotides described herein include“genes” and that the nucleic acid molecules described herein include“vectors” or “plasmids.” Accordingly, the term “gene”, also called a“structural gene” refers to a polynucleotide that codes for a particularsequence of amino acids, which comprise all or part of one or moreproteins or enzymes, and may include regulatory (non-transcribed) DNAsequences, such as promoter sequences, which determine for example theconditions under which the gene is expressed. The transcribed region ofthe gene may include untranslated regions, including introns,5′-untranslated region (UTR), and 3′-UTR, as well as the codingsequence.

The term “operon” refers to two or more genes which are transcribed as asingle transcriptional unit from a common promoter. In some embodiments,the genes comprising the operon are contiguous genes. It is understoodthat transcription of an entire operon can be modified (i.e., increased,decreased, or eliminated) by modifying the common promoter.Alternatively, any gene or combination of genes in an operon can bemodified to alter the function or activity of the encoded polypeptide.The modification can result in an increase in the activity of theencoded polypeptide. Further, the modification can impart new activitieson the encoded polypeptide. Exemplary new activities include the use ofalternative substrates and/or the ability to function in alternativeenvironmental conditions.

A “vector” is any means by which a nucleic acid can be propagated and/ortransferred between organisms, cells, or cellular components. Vectorsinclude viruses, bacteriophage, pro-viruses, plasmids, phagemids,transposons, and artificial chromosomes such as YACs (yeast artificialchromosomes), BACs (bacterial artificial chromosomes), and PLACs (plantartificial chromosomes), and the like, that are “episomes,” that is,that replicate autonomously or can integrate into a chromosome of a hostcell. A vector can also be a naked RNA polynucleotide, a naked DNApolynucleotide, a polynucleotide composed of both DNA and RNA within thesame strand, a poly-lysine-conjugated DNA or RNA, a peptide-conjugatedDNA or RNA, a liposome-conjugated DNA, or the like, that are notepisomal in nature, or it can be an organism which comprises one or moreof the above polynucleotide constructs such as an agrobacterium or abacterium.

“Transformation” refers to the process by which a vector is introducedinto a host cell. Transformation (or transduction, or transfection), canbe achieved by any one of a number of means including chemicaltransformation (e.g. lithium acetate transformation), electroporation,microinjection, biolistics (or particle bombardment-mediated delivery),or agrobacterium mediated transformation.

The term “enzyme” as used herein refers to any substance that catalyzesor promotes one or more chemical or biochemical reactions, which usuallyincludes enzymes totally or partially composed of a polypeptide, but caninclude enzymes composed of a different molecule includingpolynucleotides.

The term “protein,” “peptide,” or “polypeptide” as used herein indicatesan organic polymer composed of two or more amino acidic monomers and/oranalogs thereof. As used herein, the term “amino acid” or “amino acidicmonomer” refers to any natural and/or synthetic amino acids includingglycine and both D or L optical isomers. The term “amino acid analog”refers to an amino acid in which one or more individual atoms have beenreplaced, either with a different atom, or with a different functionalgroup. Accordingly, the term polypeptide includes amino acidic polymerof any length including full length proteins, and peptides as well asanalogs and fragments thereof. A polypeptide of three or more aminoacids is also called a protein oligomer or oligopeptide

The term “homolog,” used with respect to an original enzyme or gene of afirst family or species, refers to distinct enzymes or genes of a secondfamily or species which are determined by functional, structural orgenomic analyses to be an enzyme or gene of the second family or specieswhich corresponds to the original enzyme or gene of the first family orspecies. Most often, homologs will have functional, structural orgenomic similarities. Techniques are known by which homologs of anenzyme or gene can readily be cloned using genetic probes and PCR.Identity of cloned sequences as homolog can be confirmed usingfunctional assays and/or by genomic mapping of the genes.

A protein has “homology” or is “homologous” to a second protein if theamino acid sequence encoded by a gene has a similar amino acid sequenceto that of the second gene. Alternatively, a protein has homology to asecond protein if the two proteins have “similar” amino acid sequences.(Thus, the term “homologous proteins” is defined to mean that the twoproteins have similar amino acid sequences).

The term “analog” or “analogous” refers to nucleic acid or proteinsequences or protein structures that are related to one another infunction only and are not from common descent or do not share a commonancestral sequence. Analogs may differ in sequence but may share asimilar structure, due to convergent evolution. For example, two enzymesare analogs or analogous if the enzymes catalyze the same reaction ofconversion of a substrate to a product, are unrelated in sequence, andirrespective of whether the two enzymes are related in structure.

Recombinant Microorganisms with Reduced by-Product Accumulation

Yeast cells convert sugars to produce pyruvate, which is then utilizedin a number of pathways of cellular metabolism. In recent years, yeastcells have been engineered to produce a number of desirable products viapyruvate-driven biosynthetic pathways. In many of these biosyntheticpathways, the initial pathway step is the conversion of endogenouspyruvate to a 3-keto acid.

As used herein, a “3-keto acid” refers to an organic compound whichcontains a carboxylic acid moiety on the C1 carbon and a ketone moietyon the C3 carbon. For example, acetolactate and2-hydroxy-2-methyl-3-oxobutanoic acid are 3-keto acids with a ketonegroup at the C3 carbon (See, e.g., FIG. 2).

An example of a 3-keto acid which is common to many biosyntheticpathways is acetolactate, which is formed from pyruvate by the action ofthe enzyme acetolactate synthase (also known as acetohydroxy acidsynthase). Amongst the biosynthetic pathways using acetolactate asintermediate include pathways for the production of isobutanol,2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl,valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol,4-methyl-1-pentanol, and coenzyme A. Engineered biosynthetic pathwaysfor the synthesis of these beneficial acetolactate-derived metabolitesare found in Table 1 and FIG. 18.

TABLE 1 Biosynthetic Pathways Utilizing Acetolactate as an IntermediateBiosynthetic Pathway Reference^(a) Isobutanol US 2009/0226991 (Feldmanet al.), US 2011/0020889 (Feldman et al.), and US 2010/0143997 (Buelteret al.) 1-Butanol WO/2010/017230 (Lynch), WO/2010/031772 (Wu et al.),and KR2011002130 (Lee et al.) 2-Butanol WO/2007/130518 (Donaldson etal.), WO/2007/130521 (Donaldson et al.), and WO/2009/134276 (Donaldsonet al.) 2-Butanone WO/2007/130518 (Donaldson et al.), WO/2007/130521(Donaldson et al.), and WO/2009/134276 (Donaldson et al.) 2-3-ButanediolWO/2007/130518 (Donaldson et al.), WO/2007/130521 (Donaldson et al.),and WO/2009/134276 (Donaldson et al.) Acetoin WO/2007/130518 (Donaldsonet al.), WO/2007/130521 (Donaldson et al.), and WO/2009/134276(Donaldson et al.) Diacetyl Gonzalez et al., 2000, J. Biol. Chem 275:35876-85 and Ehsani et al., 2009, App. Environ. Micro. 75: 3196-205Valine WO/2001/021772 (Yocum et al.) and McCourt et al., 2006, AminoAcids 31: 173-210 Leucine WO/2001/021772 (Yocum et al.) and McCourt etal., 2006, Amino Acids 31: 173-210 Pantothenic Acid WO/2001/021772(Yocum et al.) 3-Methyl-1-Butanol WO/2008/098227 (Liao et al.), Atsumiet al., 2008, Nature 451: 86- 89, and Connor et al., 2008, Appl.Environ. Microbiol. 74: 5769-5775 4-Methyl-1-Pentanol WO/2010/045629(Liao et al.), Zhang et al., 2008, Proc Natl Acad Sci USA 105:20653-20658 Coenzyme A WO/2001/021772 (Yocum et al.) ^(a)The contents ofeach of the references in this table are herein incorporated byreference in their entireties for all purposes.

Each of the biosynthetic pathways listed in Table 1 shares the common3-keto acid intermediate, acetolactate. Therefore, the product yieldfrom these biosynthetic pathways will in part depend upon the amount ofacetolactate that is available to downstream enzymes of saidbiosynthetic pathways.

Another example of a 3-keto acid which is common to many biosyntheticpathways is 2-aceto-2-hydroxybutyrate, which is formed from pyruvate and2-ketobutyrate by the action of the enzyme acetolactate synthase (alsoknown as acetohydroxy acid synthase). Amongst the biosynthetic pathwaysusing 2-aceto-2-hydroxybutyrate as intermediate include pathways for theproduction of 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol,4-methyl-1-hexanol, and 5-methyl-1-heptanol. Engineered biosyntheticpathways for the synthesis of these beneficial2-aceto-2-hydroxybutyrate-derived metabolites are found in Table 2 andFIG. 19.

TABLE 2 Biosynthetic Pathways Utilizing 2-Aceto-2-Hydroxybutyrate as anIntermediate Biosynthetic Pathway Reference^(a) 2-Methyl-1-ButanolWO/2008/098227 (Liao et al.), WO/2009/076480 (Picataggio et al.), andAtsumi et al., 2008, Nature 451: 86-89 Isoleucine McCourt et al., 2006,Amino Acids 31: 173-210 3-Methyl-1-Pentanol WO/2010/045629 (Liao etal.), Zhang et al., 2008, Proc Natl Acad Sci USA 105: 20653-206584-Methyl-1-Hexanol W WO/2010/045629 (Liao et al.), Zhang et al., 2008,Proc Natl Acad Sci USA 105: 20653-20658 5-Methyl-1-HeptanolWO/2010/045629 (Liao et al.), Zhang et al., 2008, Proc Natl Acad Sci USA105: 20653-20658 ^(a)The contents of each of the references in thistable are herein incorporated by reference in their entireties for allpurposes.

Each of the biosynthetic pathways listed in Table 2 shares the common3-keto acid intermediate, 2-aceto-2-hydroxybutyrate. Therefore, theproduct yield from these biosynthetic pathways will in part depend uponthe amount of acetolactate that is available to downstream enzymes ofsaid biosynthetic pathways.

Likewise, yeast cells can be engineered to produce a number of desirableproducts via biosynthetic pathways that utilize an aldehyde as a pathwayintermediate. Engineered biosynthetic pathways comprising an aldehydeintermediate include biosynthetic pathways for the production ofisobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol,1-propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol,4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol (SeeTable 3 and FIGS. 18, 19, and 20).

TABLE 3 Biosynthetic Pathways Utilizing an Aldehyde as an IntermediateBiosynthetic Aldehyde Pathway Intermediate Reference^(a) IsobutanolIsobutyraldehyde US 2009/0226991 (Feldman et al.), US 2011/0020889(Feldman et al.), and US 2010/0143997 (Buelter et al.) 1-Butanol1-Butanal WO/2010/017230 (Lynch), WO/2010/031772 (Wu et al.),WO/2010/045629 (Liao et al.), WO/2007/041269 (Donaldson et al.),WO/2008/052991 (Raamsdonk et al.), WO/2008/143704 (Buelter et al.), andWO/2008/080124 (Gunawardena et al.) 2-Methyl-1- 2-Methyl-1-WO/2008/098227 (Liao et al.), WO/2009/076480 (Picataggio Butanol Butanalet al.), and Atsumi et al., 2008, Nature 451: 86-89 3-Methyl-1-3-Methyl-1- WO/2008/098227 (Liao et al.), Atsumi et al., 2008, NatureButanol Butanal 451: 86-89, and Connor et al., 2008, Appl. Environ.Microbiol. 74: 5769-5775 1-Propanol 1-Propanal WO/2008/098227 (Liao etal.) 1-Pentanol 1-Pentanal WO/2010/045629 (Liao et al.), Zhang et al.,2008, Proc Natl Acad Sci USA 105: 20653-20658 1-Hexanol 1-HexanalWO/2010/045629 (Liao et al.), Zhang et al., 2008, Proc Natl Acad Sci USA105: 20653-20658 3-Methyl-1- 3-Methyl-1- WO/2010/045629 (Liao et al.),Zhang et al., 2008, Proc Natl Pentanol Pentanal Acad Sci USA 105:20653-20658 4-Methyl-1- 4-Methyl-1- WO/2010/045629 (Liao et al.), Zhanget al., 2008, Proc Natl Pentanol Pentanal Acad Sci USA 105: 20653-206584-Methyl-1- 4-Methyl-1- WO/2010/045629 (Liao et al.), Zhang et al.,2008, Proc Natl Hexanol Hexanal Acad Sci USA 105: 20653-206585-Methyl-1- 5-Methyl-1- WO/2010/045629 (Liao et al.), Zhang et al.,2008, Proc Natl Heptanol Heptanal Acad Sci USA 105: 20653-20658 ^(a)Thecontents of each of the references in this table are herein incorporatedby reference in their entireties for all purposes.

Each of the biosynthetic pathways listed in Table 3 have an aldehydeintermediate. For example, the aldehyde intermediate in the isobutanolproducing metabolic pathway is isobutyraldehyde (See FIG. 1), whilepathways for the production of 1-butanol, 2-methyl-1-butanol,3-methyl-1-butanol, 1-propanol, 1-pentanol, 1-hexanol,3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol, and5-methyl-1-heptanol utilize 1-butanal, 2-methyl-1-butanal,3-methyl-1-butanal, 1-propanal, 1-pentanal, 1-hexanal,3-methyl-1-pentanal, 4-methyl-1-pentanal, 4-methyl-1-hexanal, and5-methyl-1-heptanol as aldehyde intermediates, respectively. Therefore,the product yield in biosynthetic pathways that utilize these aldehydeintermediates will in part depend upon the amount of the aldehydeintermediate that is available to downstream enzymes of saidbiosynthetic pathways.

As described herein, the present inventors have discovered the enzymaticactivities responsible for the accumulation of unwanted by-productsderived from 3-keto acid and/or aldehyde intermediates. Specifically,they have determined that a 3-ketoacid reductase and an aldehydedehydrogenase are responsible for the conversion of 3-keto acids andaldehydes, respectively, to unwanted by-products. The activities ofthese enzymes are shown to hinder the optimal productivity and yield of3-keto acid- and/or aldehyde-derived products, including, but notlimited to, those listed in Tables 1-3. The present inventors have foundthat suppressing these newly-characterized enzymatic activitiesconsiderably reduces or eliminates the formation of unwantedby-products, and concomitantly improves the yields and titers ofbeneficial metabolites.

Reduced Accumulation of 3-Hydroxyacids from 3-Keto Acids

As described herein, the present inventors have discovered that unwantedby-products, 3-hydroxyacids, can accumulate during fermentationreactions with microorganisms comprising a pathway involving a 3-ketoacid intermediate.

As used herein, a “3-hydroxyacid” is an organic compound which containsa carboxylic acid moiety on the C1 carbon and an alcohol moiety on theC3 carbon. 3-hydroxyacids can be obtained from 3-keto acids by chemicalreduction of the 3-keto acid ketone moiety to an alcohol moiety. Forexample, reduction of the ketone moiety in acetolactate or2-hydroxy-2-methyl-3-oxobutanoic acid results in the formation of3-hydroxyacid 2,3-dihydroxy-2-methylbutanoic acid (DH2MB) (See, e.g.,FIG. 2).

The present inventors have discovered that the 3-hydroxyacid by-product,2,3-dihydroxy-2-methylbutanoic acid (CAS #14868-24-7) (DH2MB),accumulates during fermentation reactions with microorganisms comprisingbiosynthetic pathways involving the 3-keto acid intermediate,acetolactate. The accumulation of this by-product was found to hinderoptimal productivity and yield of the biosynthetic pathway's targetmetabolite. The present inventors found that the production of DH2MB iscaused by the reduction of acetolactate. To reduce or eliminate theactivity responsible for the production of DH2MB, the correspondingenzymatic activity catalyzing this reaction had to be identified andreduced or eliminated. The inventors have found in S. cerevisiae thatone such enzyme catalyzing the conversion of acetolactate to DH2MB isYMR226C (also known as TMA29). This the first report of a protein inyeast that converts acetolactate to DH2MB.

The present inventors have also discovered that the 3-hydroxyacidby-product, 2-ethyl-2,3-dihydroxybutanoate, accumulates duringfermentation reactions with microorganisms comprising biosyntheticpathways involving the 3-keto acid intermediate,2-aceto-2-hydroxybutyrate. The accumulation of this by-product was foundto hinder optimal productivity and yield of the biosynthetic pathway'starget metabolite. The present inventors found that the production of2-ethyl-2,3-dihydroxybutanoate is caused by the reduction of2-aceto-2-hydroxybutyrate. To reduce or eliminate the activityresponsible for the production of 2-ethyl-2,3-dihydroxybutanoate, thecorresponding enzymatic activity catalyzing this reaction had to beidentified and reduced or eliminated. The inventors have found in S.cerevisiae, the enzyme YMR226C (also known as TMA29) which catalyzes theconversion of acetolactate to DH2MB also catalyzes the conversion of2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutanoate. This thefirst report of a protein in yeast that converts2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutanoate.

The present inventors describe herein multiple strategies for reducingthe conversion of the 3-keto acid intermediate to the corresponding3-hydroxyacid by-product, a process which is accompanied by an increasein the yield of desirable metabolites. In one embodiment, the 3-ketoacid intermediate is acetolactate and the corresponding 3-hydroxyacid isDH2MB. As described herein, reducing the conversion of acetolactate toDH2MB enables the increased production of beneficial metabolites such asisobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin,diacetyl, valine, leucine, pantothenic acid, isobutylene,3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A which arederived from biosynthetic pathways which use acetolactate as anintermediate. In another embodiment, the 3-keto acid intermediate is2-aceto-2-hydroxybutyrate and the corresponding 3-hydroxyacid is2-ethyl-2,3-dihydroxybutanoate. As described herein, reducing theconversion of 2-aceto-2-hydroxybutyrate to2-ethyl-2,3-dihydroxybutanoate enables the increased production ofbeneficial metabolites such as 2-methyl-1-butanol, isoleucine,3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.

Accordingly, one aspect of the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses a 3-keto acidas an intermediate, wherein said recombinant microorganism issubstantially free of an enzyme that catalyzes the conversion of the3-keto acid intermediate to a 3-hydroxyacid by-product. In oneembodiment, the 3-keto acid intermediate is acetolactate and the3-hydroxyacid by-product is DH2MB. In another embodiment, the 3-ketoacid intermediate is 2-aceto-2-hydroxybutyrate and the 3-hydroxyacidby-product is 2-ethyl-2,3-dihydroxybutanoate.

In another aspect, the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses a 3-keto acidas an intermediate, wherein said recombinant microorganism is engineeredto reduce or eliminate the expression or activity of an enzymecatalyzing the conversion of the 3-keto acid intermediate to a3-hydroxyacid by-product. In one embodiment, the 3-keto acidintermediate is acetolactate and the 3-hydroxyacid by-product is DH2MB.In another embodiment, the 3-keto acid intermediate is2-aceto-2-hydroxybutyrate and the 3-hydroxyacid by-product is2-ethyl-2,3-dihydroxybutanoate.

In various embodiments described herein, the protein involved incatalyzing the conversion of the 3-keto acid intermediate to the3-hydroxyacid by-product is a ketoreductase. In an exemplary embodiment,the ketoreductase is a 3-ketoacid reductase (3-KAR). As used herein, theterm “3-ketoacid reductase” refers to a ketoreductase (i.e. ketonereductase) active towards the 3-oxo group of a 3-keto acid. Anillustration of exemplary reactions capable of being catalyzed by3-ketoacid reductases is shown in FIG. 2. Suitable 3-ketoacid reductasesare generally found in the enzyme classification subgroup 1.1.1.X, thefinal digit X being dependent upon the substrate. A non-limiting list ofexemplary 3-ketoacid reductases and their corresponding enzymeclassification number is shown in FIG. 3.

In an exemplary embodiment, the 3-ketoacid reductase is the S.cerevisiae YMR226C (SEQ ID NO: 1) protein, used interchangeably hereinwith “TMA29”. In some embodiments, the 3-ketoacid reductase is the S.cerevisiae YMR226C (SEQ ID NO: 1) protein or a homolog or variantthereof. In one embodiment, the homolog may be selected from the groupconsisting of Vanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomycescastellii (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomycesbayanus (SEQ ID NO: 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), K.lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyceskluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQ ID NO: 10),Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12),Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14),Candida dubliniensis (SEQ ID NO: 15), Candida albicans (SEQ ID NO: 16),Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO:18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO:20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).

In one embodiment, the recombinant microorganism of the inventionincludes a mutation in at least one gene encoding for a 3-ketoacidreductase resulting in a reduction of 3-ketoacid reductase activity of apolypeptide encoded by said gene. In another embodiment, the recombinantmicroorganism includes a partial deletion of a gene encoding for a3-ketoacid reductase gene resulting in a reduction of 3-ketoacidreductase activity of a polypeptide encoded by the gene. In anotherembodiment, the recombinant microorganism comprises a complete deletionof a gene encoding for a 3-ketoacid reductase resulting in a reductionof 3-ketoacid reductase activity of a polypeptide encoded by the gene.In yet another embodiment, the recombinant microorganism includes amodification of the regulatory region associated with the gene encodingfor a 3-ketoacid reductase resulting in a reduction of expression of apolypeptide encoded by said gene. In yet another embodiment, therecombinant microorganism comprises a modification of thetranscriptional regulator resulting in a reduction of transcription ofgene encoding for a 3-ketoacid reductase. In yet another embodiment, therecombinant microorganism comprises mutations in all genes encoding fora 3-ketoacid reductase resulting in a reduction of activity of apolypeptide encoded by the gene(s). In one embodiment, said 3-ketoacidreductase gene is the S. cerevisiae TMA29 (YMR226C) gene or a homologthereof. As would be understood in the art, naturally occurring homologsof TMA29 in yeast other than S. cerevisiae can similarly be inactivatedusing the methods of the present invention. TMA29 homologs and methodsof identifying such TMA29 homologs are described herein.

As is understood by those skilled in the art, there are severaladditional mechanisms available for reducing or disrupting the activityof a protein such as 3-ketoacid reductase, including, but not limitedto, the use of a regulated promoter, use of a weak constitutivepromoter, disruption of one of the two copies of the gene in a diploidyeast, disruption of both copies of the gene in a diploid yeast,expression of an anti-sense nucleic acid, expression of an siRNA, overexpression of a negative regulator of the endogenous promoter,alteration of the activity of an endogenous or heterologous gene, use ofa heterologous gene with lower specific activity, the like orcombinations thereof.

As described herein, the recombinant microorganisms of the presentinvention are engineered to produce less of the 3-hydroxyacid by-productthan an unmodified parental microorganism. In one embodiment, therecombinant microorganism produces the 3-hydroxyacid by-product from acarbon source at a carbon yield of less than about 20 percent. Inanother embodiment, the microorganism is produces the 3-hydroxyacidby-product from a carbon source at a carbon yield of less than about 10,less than about 5, less than about 2, less than about 1, less than about0.5, less than about 0.1, or less than about 0.01 percent. In oneembodiment, the 3-hydroxyacid by-product is DH2MB, derived from the3-keto acid, acetolactate. In another embodiment, the 3-hydroxyacidby-product is 2-ethyl-2,3-dihydroxybutanoate, derived from the 3-ketoacid, 2-aceto-2-hydroxybutyrate.

In one embodiment, the 3-hydroxyacid by-product carbon yield derivedfrom the 3-ketoacid is reduced by at least about 50% in a recombinantmicroorganism as compared to a parental microorganism that does notcomprise a reduction or deletion of the activity or expression of one ormore endogenous proteins involved in catalyzing the conversion of the3-ketoacid intermediate to the 3-hydroxyacid by-product. In anotherembodiment, the 3-hydroxyacid by-product derived from the 3-ketoacid isreduced by at least about 60%, by at least about 65%, by at least about70%, by at least about 75%, by at least about 80%, by at least about85%, by at least about 90%, by at least about 95%, by at least about99%, by at least about 99.9%, or by at least about 100% as compared to aparental microorganism that does not comprise a reduction or deletion ofthe activity or expression of one or more endogenous proteins involvedin catalyzing the conversion of the 3-ketoacid to the 3-hydroxyacidby-product. In one embodiment, the 3-hydroxyacid by-product is DH2MB,derived from the 3-keto acid, acetolactate.

In another embodiment, the 3-hydroxyacid by-product is2-ethyl-2,3-dihydroxybutanoate, derived from the 3-keto acid,2-aceto-2-hydroxybutyrate. In an additional embodiment, the yield of adesirable fermentation product is increased in the recombinantmicroorganisms comprising a reduction or elimination of the activity orexpression of one or more endogenous proteins involved in catalyzing theconversion of the 3-ketoacid intermediate to the 3-hydroxyacidby-product. In one embodiment, the yield of a desirable fermentationproduct is increased by at least about 1% as compared to a parentalmicroorganism that does not comprise a reduction or elimination of theactivity or expression of one or more endogenous proteins involved incatalyzing the conversion of the 3-ketoacid intermediate to the3-hydroxyacid by-product. In another embodiment, the yield of adesirable fermentation product is increased by at least about 5%, by atleast about 10%, by at least about 25%, or by at least about 50% ascompared to a parental microorganism that does not comprise a reductionor elimination of the activity or expression of one or more endogenousproteins involved in catalyzing the conversion of the 3-ketoacidintermediate to the 3-hydroxyacid by-product. In one embodiment, the3-hydroxyacid by-product is DH2MB, derived from the 3-keto acid,acetolactate. Accordingly, in one embodiment, the desirable fermentationproduct is derived from any biosynthetic pathway in which acetolactateacts as an intermediate, including, but not limited to, isobutanol,2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl,valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol,4-methyl-1-pentanol, and coenzyme A. In another embodiment, the3-hydroxyacid by-product is 2-ethyl-2,3-dihydroxybutanoate, derived fromthe 3-keto acid, 2-aceto-2-hydroxybutyrate. Accordingly, in anotherembodiment, the desirable fermentation product is derived from anybiosynthetic pathway in which 2-aceto-2-hydroxybutyrate acts as anintermediate, including, but not limited to, 2-methyl-1-butanol,isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and5-methyl-1-heptanol.

In further embodiments, additional enzymes potentially catalyzing theconversion of a 3-ketoacid intermediate to a 3-hydroxyacid by-productare deleted from the genome of a recombinant microorganism comprising abiosynthetic pathway which uses a 3-ketoacid as an intermediate.Endogenous yeast genes with the potential to convert of a 3-ketoacidintermediate to a 3-hydroxyacid by-product include ketoreductases, shortchain alcohol dehydrogenases, medium chain alcohol dehydrogenases,members of the aldose reductase family, members of the D-hydroxyaciddehydrogenase family, alcohol dehydrogenases, and lactatedehydrogenases. In one embodiment, the 3-hydroxyacid by-product isDH2MB, derived from the 3-keto acid, acetolactate. In anotherembodiment, the 3-hydroxyacid by-product is2-ethyl-2,3-dihydroxybutanoate, derived from the 3-keto acid,2-aceto-2-hydroxybutyrate.

Methods for identifying additional enzymes catalyzing the conversion ofa 3-ketoacid intermediate to a 3-hydroxyacid by-product are outlined asfollows: endogenous yeast genes coding for ketoreductases, short chainalcohol dehydrogenases, medium chain alcohol dehydrogenases, members ofthe aldose reductase family, members of the D-hydroxyacid dehydrogenasefamily, alcohol dehydrogenases, and lactate dehydrogenases are deletedfrom the genome of a yeast strain comprising a biosynthetic pathway inwhich a 3-ketoacid (e.g., acetolactate or 2-aceto-2-hydroxybutyrate) isan intermediate. These deletion strains are compared to the parentstrain by fermentation and analysis of the fermentation broth for thepresence and concentration of the corresponding 3-hydroxyacid by-product(e.g., DH2MB or 2-ethyl-2,3-dihydroxybutanoate, derived fromacetolactate and 2-aceto-2-hydroxybutyrate, respectively). In S.cerevisiae, deletions that reduce the production of the 3-hydroxyacidby-product are combined by construction of strains carrying multipledeletions. Candidate genes can include, but are not limited to, YAL060W,YJR159W, YGL157W, YBL114W, YOR120W, YKL055C, YBR159W, YBR149W, YDL168W,YDR368W, YLR426W, YCR107W, YILL24W, YML054C, YOL151W, YMR318C, YBR046C,YHR104W, YIR036C, YDL174C, YDR541C, YBR145W, YGL039W, YCR105W, YDL124W,YIR035C, YFL056C, YNL274c, YLR255C, YGL185C, YGL256W, YJR096W, YJR155W,YPL275W, YOR388C, YLR070C, YMR083W, YER081W, YJR139C, YDL243C, YPL113C,YOL165C, YML086C, YMR303C, YDL246C, YLR070C, YHR063C, YNL331C, YFL057C,YIL155C, YOL086C, YAL061W, YDR127W, YPR127W, YCL018W, YIL074C, YIL124W,and YEL071W. Many of these deletion strains are available commercially(for example Open Biosystems YSC1054). These deletion strains aretransformed with a plasmid pGV2435 from which the ALS gene (e.g., the B.subtilis alsS) is expressed under the control of the CUP1 promoter. Thetransformants are cultivated in YPD medium containing 150 g/L glucose inshake flasks at 30° C., 75 rpm in a shaking incubator for 48 hours.After 48 h samples from the shake flasks are analyzed by HPLC for theconcentration of the 3-hydroxyacid by-product (e.g., DH2MB and2-ethyl-2,3-dihydroxybutanoate, derived from acetolactate and2-aceto-2-hydroxybutyrate, respectively). As would be understood in theart, naturally occurring homologs of 3-ketoacid reductase genes (e.g.,TMA29) in yeast other than S. cerevisiae can similarly be inactivated.3-ketoacid reductase gene (e.g., TMA29) homologs and methods ofidentifying such 3-ketoacid reductase gene homologs are describedherein.

Another way to screen the deletion library is to incubate yeast cellswith the 3-ketoacid intermediate (e.g., acetolactate or2-aceto-2-hydroxybutyrate) and analyze the broth for the production ofthe corresponding 3-hydroxyacid by-product (e.g., DH2MB or2-ethyl-2,3-dihydroxybutanoate, derived from acetolactate and2-aceto-2-hydroxybutyrate, respectively).

Some of the listed genes are the result of tandem duplication or wholegenome duplication events and are expected to have similar substratespecificities. Examples are YAL061W (BDH1), and YAL060W (BDH2), YDR368W(YPR1) and YOR120W (GCY1). Deletion of just one of the duplicated genesis likely not to result in a phenotype. These gene pairs have to beanalyzed in strains carrying deletions in both genes.

An alternative approach to find additional endogenous activityresponsible for the production of the 3-hydroxyacid by-product (e.g.,DH2MB or 2-ethyl-2,3-dihydroxybutanoate, derived from acetolactate and2-aceto-2-hydroxybutyrate, respectively) is to analyze yeast strainsthat overexpress the genes suspected of encoding the enzyme responsiblefor production of the 3-hydroxyacid by-product. Such strains arecommercially available for many of the candidate genes listed above (forexample Open Biosystems YSC3870). The ORF overexpressing strains areprocessed in the same way as the deletion strains. They are transformedwith a plasmid for ALS expression and screened for 3-hydroxyacidby-product (e.g., DH2MB or 2-ethyl-2,3-dihydroxybutanoate) productionlevels. To narrow the list of possible genes causing the production ofthe 3-hydroxyacid by-product (e.g., DH2MB or2-ethyl-2,3-dihydroxybutanoate), their expression can be analyzed infermentation samples. Genes that are not expressed during a fermentationthat produced the 3-hydroxyacid by-product (e.g., DH2MB or2-ethyl-2,3-dihydroxybutanoate) can be excluded from the list ofpossible targets. This analysis can be done by extraction of RNA fromfermenter samples and submitting these samples to whole genomeexpression analysis, for example, by Roche NimbleGen.

As described herein, strains that naturally produce low levels of one ormore 3-hydroxyacid by-products can also have applicability for producingincreased levels of desirable fermentation products that are derivedfrom biosynthetic pathways comprising a 3-ketoacid intermediate. Aswould be understood by one skilled in the art equipped with the instantdisclosure, strains that naturally produce low levels of one or more3-hydroxyacid by-products may inherently exhibit low or undetectablelevels of endogenous enzyme activity, resulting in the reducedconversion of 3-ketoacids to 3-hydroxyacids, a trait favorable for theproduction of a desirable fermentation product such as isobutanol.Described herein are several approaches for identifying a native hostmicroorganism which is substantially free of 3-ketoacid reductaseactivity. For example, one approach to finding a host microorganismwhich exhibits inherently low or undetectable endogenous enzyme activityresponsible for the production of the 3-hydroxyacid by-product (e.g.,DH2MB or 2-ethyl-2,3-dihydroxybutanoate) is to analyze yeast strains byincubating the yeast cells with a 3-keto acid (e.g., acetolactate or2-aceto-2-hydroxybutyrate) and analyze the broth for the production ofthe corresponding 3-hydroxyacid by-product (e.g., DH2MB or2-ethyl-2,3-dihydroxybutanoate, derived from acetolactate and2-aceto-2-hydroxybutyrate, respectively).

The recombinant microorganisms described herein which produce abeneficial metabolite derived from a biosynthetic pathway which uses a3-keto acid as an intermediate may be further engineered to reduce oreliminate enzymatic activity for the conversion of pyruvate to productsother than the 3-keto acid (e.g., acetolactate and/or2-aceto-2-hydroxybutyrate). In one embodiment, the enzymatic activity ofpyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvateoxidase, pyruvate dehydrogenase, and/or glycerol-3-phosphatedehydrogenase (GPD) is reduced or eliminated.

In a specific embodiment, the beneficial metabolite is produced in arecombinant PDC-minus GPD-minus yeast microorganism that overexpressesan acetolactate synthase (ALS) gene. In another specific embodiment, theALS is encoded by the B. subtilis alsS.

Reduced Accumulation of Acid by-Products from Aldehyde Intermediates

As described further in the Examples, the present inventors have alsodiscovered that unwanted acid by-products (e.g., isobutyrate in the caseof isobutanol), can accumulate during fermentation reactions withmicroorganisms comprising a pathway involving an aldehyde intermediate(e.g., isobutyraldehyde in the case of isobutanol).

As used herein, an “acid by-product” refers to an organic compound whichcontains a carboxylic acid moiety. An acid by-product can be obtained bythe oxidation of an aldehyde. For example, the oxidation ofisobutyraldehyde results in the formation of isobutyric acid (See, e.g.,FIG. 4).

The present inventors have found that accumulation of these acidby-products hinders the optimal productivity and yield of thebiosynthetic pathway which utilize aldehyde intermediates. The presentinventors found that the production of these acid by-products is causedby dehydrogenation of the corresponding aldehyde. To reduce or eliminatethe activity responsible for the production of the acid by-product, thecorresponding enzymatic activity catalyzing this reaction had to beidentified and reduced or eliminated. The inventors have found in S.cerevisiae that one such enzyme catalyzing the conversion of aldehydesto acid by-products is aldehyde dehydrogenase.

The present inventors describe herein multiple strategies for reducingacid by-product formation, a process which is accompanied by an increasein the yield of desirable metabolites such as isobutanol, 1-butanol,2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol, 1-pentanol,1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol,and 5-methyl-1-heptanol.

Accordingly, one aspect of the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses an aldehydeas an intermediate, wherein said recombinant microorganism issubstantially free of an enzyme that catalyzes the conversion of analdehyde to an acid by-product.

In another aspect, the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses an aldehydeas an intermediate, wherein said recombinant microorganism is engineeredto reduce or eliminate the expression or activity of one or more enzymescatalyzing the conversion of the aldehyde to an acid by-product.

In one embodiment, the aldehyde intermediate is isobutyraldehyde and theacid by-product is isobutyrate. In another embodiment, the aldehydeintermediate is 1-butanal and the acid by-product is butyrate. In yetanother embodiment, the aldehyde intermediate is 2-methyl-1-butanal andthe acid by-product is 2-methyl-1-butyrate. In yet another embodiment,the aldehyde intermediate is 3-methyl-1-butanal and the acid by-productis 3-methyl-1-butyrate. In yet another embodiment, the aldehydeintermediate is 1-propanal and the acid by-product is propionate. In yetanother embodiment, the aldehyde intermediate is 1-pentanal and the acidby-product is pentanoate. In yet another embodiment, the aldehydeintermediate is 1-hexanal and the acid by-product is hexanoate. In yetanother embodiment, the aldehyde intermediate is 3-methyl-1-pentanal andthe acid by-product is 3-methyl-1-pentanoate. In yet another embodiment,the aldehyde intermediate is 4-methyl-1-pentanal and the acid by-productis 4-methyl-1-pentanoate. In yet another embodiment, the aldehydeintermediate is 4-methyl-1-hexanal and the acid by-product is4-methyl-1-hexanoate. In yet another embodiment, the aldehydeintermediate is 5-methyl-1-heptanal and the acid by-product is5-methyl-1-heptanoate.

In various embodiments described herein, the protein involved incatalyzing the conversion of an aldehyde to acid by-product is analdehyde dehydrogenase (ALDH).

As used herein, the term “aldehyde dehydrogenase” refers to an enzymecatalyzing the reaction:

an aldehyde+oxidized cofactor+H₂O=an acid+reduced cofactor+H⁺

An illustration of exemplary reactions capable of being catalyzed byaldehyde dehydrogenases is shown in FIG. 4. Suitable aldehydedehydrogenases are generally found in the enzyme classification subgroupEC 1.2.1.X, wherein the final digit X is dependent upon the substrate orthe cofactor. For example, EC 1.2.1.3 catalyzes the following reaction:an aldehyde+NAD⁺+H₂O=an acid+NADH+H⁺); EC 1.2.1.4 catalyzes thefollowing reaction: an aldehyde+NADP⁺+H₂O=an acid+NADPH+H⁺); andEC1.2.1.5 catalyzes the following reaction: an aldehyde+NAD(P)⁺+H₂O=anacid+NAD(P)H+H⁺.

As described herein, the protein involved in catalyzing the conversionof an aldehyde to an acid by-product is an aldehyde dehydrogenase(ALDH). In one embodiment, the aldehyde dehydrogenase is encoded by agene selected from the group consisting of ALD2, ALD3, ALD4, ALD5, ALD6,and HFD1, and homologs and variants thereof. In an exemplary embodiment,the aldehyde dehydrogenase is the S. cerevisiae aldehyde dehydrogenaseALD6 (SEQ ID NO: 25) or a homolog or variant thereof. In one embodiment,the homolog may be selected from the group consisting of Saccharomycescastelli (SEQ ID NO: 26), Candida glabrata (SEQ ID NO: 27),Saccharomyces bayanus (SEQ ID NO: 28), Kluyveromyces lactis (SEQ ID NO:29), Kluyveromyces thermotolerans (SEQ ID NO: 30), Kluyveromyces waltii(SEQ ID NO: 31), Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32),Saccharomyces cerevisiae JAY291 (SEQ ID NO: 33), Saccharomycescerevisiae EC1118 (SEQ ID NO: 34), Saccharomyces cerevisiae DBY939 (SEQID NO: 35), Saccharomyces cerevisiae AWR11631 (SEQ ID NO: 36),Saccharomyces cerevisiae RM11-1a (SEQ ID NO: 37), Pichia pastoris (SEQID NO: 38), Kluyveromyces marxianus (SEQ ID NO: 39), Schizosaccharomycespombe (SEQ ID NO: 40), and Schizosaccharomyces pombe (SEQ ID NO: 41).

In one embodiment, the recombinant microorganism includes a mutation inat least one gene encoding for an aldehyde dehydrogenase resulting in areduction of aldehyde dehydrogenase activity of a polypeptide encoded bysaid gene. In another embodiment, the recombinant microorganism includesa partial deletion of gene encoding for an aldehyde dehydrogenaseresulting in a reduction of aldehyde dehydrogenase activity of apolypeptide encoded by the gene. In another embodiment, the recombinantmicroorganism comprises a complete deletion of a gene encoding for analdehyde dehydrogenase resulting in a reduction of aldehydedehydrogenase activity of a polypeptide encoded by the gene. In yetanother embodiment, the recombinant microorganism includes amodification of the regulatory region associated with the gene encodingfor an aldehyde dehydrogenase resulting in a reduction of expression ofa polypeptide encoded by said gene. In yet another embodiment, therecombinant microorganism comprises a modification of thetranscriptional regulator resulting in a reduction of transcription of agene encoding for an aldehyde dehydrogenase. In yet another embodiment,the recombinant microorganism comprises mutations in all genes encodingfor an aldehyde dehydrogenase resulting in a reduction of activity of apolypeptide encoded by the gene(s). In one embodiment, said aldehydedehydrogenase is encoded by a gene selected from the group consisting ofALD2, ALD3, ALD4, ALD5, ALD6, and HFD1, and homologs and variantsthereof. As would be understood in the art, naturally occurring homologsof aldehyde dehydrogenase in yeast other than S. cerevisiae cansimilarly be inactivated using the methods of the present invention.Aldehyde dehydrogenase homologs and methods of identifying such aldehydedehydrogenase homologs are described herein.

As is understood by those skilled in the art, there are severaladditional mechanisms available for reducing or disrupting the activityof a protein such as aldehyde dehydrogenase, including, but not limitedto, the use of a regulated promoter, use of a weak constitutivepromoter, disruption of one of the two copies of the gene in a diploidyeast, disruption of both copies of the gene in a diploid yeast,expression of an anti-sense nucleic acid, expression of an siRNA, overexpression of a negative regulator of the endogenous promoter,alteration of the activity of an endogenous or heterologous gene, use ofa heterologous gene with lower specific activity, the like orcombinations thereof.

As would be understood by one skilled in the art, the activity orexpression of more than one aldehyde dehydrogenase can be reduced oreliminated. In one specific embodiment, the activity or expression ofALD4 and ALD6 or homologs or variants thereof is reduced or eliminated.In another specific embodiment, the activity or expression of ALD5 andALD6 or homologs or variants thereof is reduced or eliminated. In yetanother specific embodiment, the activity or expression of ALD4, ALD5,and ALD6 or homologs or variants thereof is reduced or eliminated. Inyet another specific embodiment, the activity or expression of thecytosolically localized aldehyde dehydrogenases ALD2, ALD3, and ALD6 orhomologs or variants thereof is reduced or eliminated. In yet anotherspecific embodiment, the activity or expression of the mitochondriallylocalized aldehyde dehydrogenases, ALD4 and ALD5 or homologs or variantsthereof, is reduced or eliminated.

As described herein, the recombinant microorganisms of the presentinvention are engineered to produce less of the acid by-product than anunmodified parental microorganism. In one embodiment, the recombinantmicroorganism produces the acid by-product from a carbon source at acarbon yield of less than about 50 percent as compared to a parentalmicroorganism. In another embodiment, the microorganism is produces theacid by-product from a carbon source from a carbon source at a carbonyield of less than about 25, less than about 10, less than about 5, lessthan about 1, less than about 0.5, less than about 0.1, or less thanabout 0.01 percent as compared to a parental microorganism. In oneembodiment, the acid by-product is isobutyrate, derived fromisobutyraldehyde, an intermediate of the isobutanol biosyntheticpathway. In another embodiment, the acid by-product is butyrate, derivedfrom 1-butanal, an intermediate of the 1-butanol biosynthetic pathway.In yet another embodiment, the acid by-product is 2-methyl-1-butyrate,derived from 2-methyl-1-butanal, an intermediate of the2-methyl-1-butanol biosynthetic pathway. In yet another embodiment, theacid by-product is 3-methyl-1-butyrate, derived from 3-methyl-1-butanal,an intermediate of the 3-methyl-1-butanol biosynthetic pathway. In yetanother embodiment, the acid by-product is propionate, derived from1-propanal, an intermediate of the 1-propanol biosynthetic pathway. Inyet another embodiment, the acid by-product is pentanoate, derived from1-pentanal, an intermediate of the 1-pentanol biosynthetic pathway. Inyet another embodiment, the acid by-product is hexanoate, derived from1-hexanal, an intermediate of the 1-hexanol biosynthetic pathway. In yetanother embodiment, the acid by-product is 3-methyl-1-pentanoate,derived from 3-methyl-1-pentanal, an intermediate of the3-methyl-1-pentanol biosynthetic pathway. In yet another embodiment, theacid by-product is 4-methyl-1-pentanoate, derived from4-methyl-1-pentanal, an intermediate of the 4-methyl-1-pentanolbiosynthetic pathway. In yet another embodiment, the acid by-product is4-methyl-1-hexanoate, derived from 4-methyl-1-hexanal, an intermediateof the 4-methyl-1-hexanol biosynthetic pathway. In yet anotherembodiment, the acid by-product is 5-methyl-1-heptanoate, derived from5-methyl-1-heptanal, an intermediate of the 5-methyl-1-heptanolbiosynthetic pathway.

In one embodiment, the acid by-product carbon yield from thecorresponding aldehyde is reduced by at least about 50% in a recombinantmicroorganism as compared to a parental microorganism that does notcomprise a reduction or deletion of the activity or expression of one ormore proteins involved in catalyzing the conversion of an aldehyde to anacid by-product. In another embodiment, the acid by-product carbon yieldfrom acetolactate is reduced by at least about 60%, by at least about65%, by at least about 70%, by at least about 75%, by at least about80%, by at least about 85%, by at least about 90%, by at least about95%, by at least about 99%, by at least about 99.9%, or by at leastabout 100% as compared to a parental microorganism that does notcomprise a reduction or deletion of the activity or expression of one ormore proteins involved in catalyzing the conversion of an aldehyde to anacid by-product. In one embodiment, the acid by-product is isobutyrate,derived from isobutyraldehyde, an intermediate of the isobutanolbiosynthetic pathway. In another embodiment, the acid by-product isbutyrate, derived from 1-butanal, an intermediate of the 1-butanolbiosynthetic pathway. In yet another embodiment, the acid by-product is2-methyl-1-butyrate, derived from 2-methyl-1-butanal, an intermediate ofthe 2-methyl-1-butanol biosynthetic pathway. In yet another embodiment,the acid by-product is 3-methyl-1-butyrate, derived from3-methyl-1-butanal, an intermediate of the 3-methyl-1-butanolbiosynthetic pathway. In yet another embodiment, the acid by-product ispropionate, derived from 1-propanal, an intermediate of the 1-propanolbiosynthetic pathway. In yet another embodiment, the acid by-product ispentanoate, derived from 1-pentanal, an intermediate of the 1-pentanolbiosynthetic pathway. In yet another embodiment, the acid by-product ishexanoate, derived from 1-hexanal, an intermediate of the 1-hexanolbiosynthetic pathway. In yet another embodiment, the acid by-product is3-methyl-1-pentanoate, derived from 3-methyl-1-pentanal, an intermediateof the 3-methyl-1-pentanol biosynthetic pathway. In yet anotherembodiment, the acid by-product is 4-methyl-1-pentanoate, derived from4-methyl-1-pentanal, an intermediate of the 4-methyl-1-pentanolbiosynthetic pathway. In yet another embodiment, the acid by-product is4-methyl-1-hexanoate, derived from 4-methyl-1-hexanal, an intermediateof the 4-methyl-1-hexanol biosynthetic pathway. In yet anotherembodiment, the acid by-product is 5-methyl-1-heptanoate, derived from5-methyl-1-heptanal, an intermediate of the 5-methyl-1-heptanolbiosynthetic pathway.

In an additional embodiment, the yield of a desirable fermentationproduct is increased in the recombinant microorganisms comprising areduction or elimination of the activity or expression of one or moreproteins involved in catalyzing the conversion of an aldehyde to acidby-product. In one embodiment, the yield of a desirable fermentationproduct is increased by at least about 1% as compared to a parentalmicroorganism that does not comprise a reduction or elimination of theactivity or expression of one or more endogenous proteins involved incatalyzing the conversion of an aldehyde to acid by-product. In anotherembodiment, the yield of a desirable fermentation product is increasedby at least about 5%, by at least about 10%, by at least about 25%, orby at least about 50% as compared to a parental microorganism that doesnot comprise a reduction or elimination of the activity or expression ofone or more endogenous proteins involved in catalyzing the conversion ofan aldehyde to acid by-product. As described herein, the desirablefermentation product may be derived from any biosynthetic pathway inwhich an aldehyde acts as an intermediate, including, but not limitedto, isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol,1-propanol, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol,4-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanolbiosynthetic pathways.

Methods for identifying additional enzymes catalyzing the conversion ofan aldehyde to acid by-product are outlined as follows: endogenous yeastgenes coding for putative aldehyde and alcohol dehydrogenases aredeleted from the genome of a yeast strain. These deletion strains arecompared to the parent strain by enzymatic assay. Many of these deletionstrains are available commercially (for example Open BiosystemsYSC1054).

Another way to screen the deletion library is to incubate yeast cellswith an aldehyde (e.g., isobutyraldehyde or 1-butanal) and analyze thebroth for the production of the corresponding acid by-product (e.g.,isobutyrate or butyrate, derived from isobutyraldehyde or 1-butanal,respectively).

An alternative approach to find additional endogenous activityresponsible for the production of the acid by-product (e.g., isobutyrateor butyrate, derived from isobutyraldehyde or 1-butanal, respectively)is to analyze yeast strains that overexpress the genes suspected ofencoding the enzyme responsible for production of the acid by-product.Such strains are commercially available for many of the candidate geneslisted above (for example Open Biosystems YSC3870). The ORFoverexpressing strains are screened for increased acid by-productproduction levels. Alternatively, the cell lysates of the ORFoverexpressing strains are assayed for increased aldehyde oxidationactivity. To narrow the list of possible genes causing the production ofacid by-products, their expression can be analyzed in fermentationsamples. Genes that are not expressed during a fermentation thatproduces an acid by-product can be excluded from the list of possibletargets. This analysis can be done by extraction of RNA from fermentersamples and submitting these samples to whole genome expressionanalysis, for example, by Roche NimbleGen.

As described herein, strains that naturally produce low levels of one ormore acid by-products can also have applicability for producingincreased levels of desirable fermentation products that are derivedfrom biosynthetic pathways comprising an aldehyde intermediate. As wouldbe understood by one skilled in the art equipped with the instantdisclosure, strains that naturally produce low levels of one or moreacid by-products may inherently exhibit low or undetectable levels ofendogenous enzyme activity, resulting in the reduced conversion ofaldehydes to acid by-products, a trait favorable for the production of adesirable fermentation product such as isobutanol. Described herein areseveral approaches for identifying a native host microorganism which issubstantially free of aldehyde dehydrogenase activity. For example, oneapproach to finding a host microorganism which exhibits inherently lowor undetectable endogenous enzyme activity responsible for theproduction of the acid by-product (e.g., isobutyrate or butyrate) is toanalyze yeast strains by incubating the yeast cells with an aldehyde(e.g., isobutyraldehyde or 1-butanal) and analyze the broth for theproduction of the corresponding acid by-product (e.g., isobutyrate orbutyrate, derived from isobutyraldehyde or 1-butanal, respectively).

As described above, one strategy reducing the production of the acidby-product, isobutyrate, is to reduce or eliminate the activity orexpression of one or more endogenous aldehyde dehydrogenase proteinspresent in yeast that may be converting isobutyraldehyde to isobutyrate.

Another strategy for reducing the production of isobutyrate is thereduction or elimination of activity or expression of one moreendogenous yeast alcohol dehydrogenases. Reducing the expression of ordeleting one or more alcohol dehydrogenases including, but not limitedto, S. cerevisiae ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7, and SFA1,and homologs or variants thereof, will generally lead to a reducedproduction of isobutyrate and a concomitant increase in isobutanolyield. The reduction and/or deletion of additional dehydrogenases areenvisioned herein and are considered within the scope of the presentinvention. These dehydrogenases include additional alcoholdehydrogenases such as S. cerevisiae BDH1, BDH2, SOR1, SOR2, and XYL1,and homologs or variants thereof, as well as aryl alcohol dehydrogenasessuch as AAD3, AAD4, AAD6, AAD10, AAD14, AAD15, AAD16, and YPL088W, andhomologs or variants thereof.

In another embodiment, the invention provides recombinant microorganismsengineered to reduce and/or deletion one or more additional genesencoding carbonyl/aldehyde reductases. These carbonyl/aldehydereductases include S. cerevisiae ARI1, YPR1, TMA29, YGL039W, and UGA2,and homologs or variants thereof.

An additional strategy described herein for reducing the production ofthe by-product isobutyrate is to reduce or eliminate the activity orexpression of endogenous proteins present in yeast that may be producingisobutyrate from the isobutanol pathway intermediate 2-ketoisovalerate.Such enzymes are generally referred to as ketoacid dehydrogenases (KDH).Elimination or reduction of the activity or expression of theseendogenous proteins can reduce or eliminate the production of theunwanted byproduct, isobutyrate. KDH enzyme activity has been identifiedin S. cerevisiae (Dickinson, J. R., and I. W. Dawes, 1992, Thecatabolism of branched-chain amino acids occurs via a 2-oxoaciddehydrogenase in S. cerevisiae. J. Gen. Microbiol. 138: 2029-2033).Reducing the expression of or deleting one or more ketoaciddehydrogenases and homologs or variants thereof, will generally lead toa reduced production of isobutyrate and a concomitant increase inisobutanol yield.

The reduction in expression of or deletion of genes in S. cerevisiae andother yeast can be achieved by methods known to those of skill in theart, such as allelic replacement or exchange, as well as gene disruptionby the insertion of another gene or marker cassette.

Another strategy described herein for reducing the production of theby-product isobutyrate is to increase the activity and/or expression ofan alcohol dehydrogenase (ADH) responsible for the conversion ofisobutyraldehyde to isobutanol. This strategy prevents competition byendogenous enzymes for the isobutanol pathway intermediate,isobutyraldehyde. An increase in the activity and/or expression of thealcohol dehydrogenase may be achieved by various means. For example,alcohol dehydrogenase activity can be increased by utilizing a promoterwith increased promoter strength, by increasing the copy number of thealcohol dehydrogenase gene, or by utilizing an alternative or modifiedalcohol dehydrogenase with increased specific activity.

An alternative strategy described herein for reducing the production ofthe by-product isobutyrate is to utilize an alcohol dehydrogenase (ADH)in the isobutanol pathway responsible for the conversion ofisobutyraldehyde to isobutanol which exhibits a decrease inMichaelis-Menten constant (K_(M)). This strategy also preventscompetition by endogenous enzymes for the isobutanol pathwayintermediate, isobutyraldehyde.

Another strategy described herein for reducing the production of theby-product isobutyrate is to utilize an alcohol dehydrogenase (ADH) inthe isobutanol pathway responsible for the conversion ofisobutyraldehyde to isobutanol which exhibits increased activity and adecrease in Michaelis-Menten constant (K_(M)). This strategy alsoprevents competition by endogenous enzymes for the isobutanol pathwayintermediate, isobutyraldehyde.

Further, by utilizing a modified ADH enzyme, the present inventors mayestablish a situation in which the forward reaction (i.e. theisobutyraldehyde conversion to isobutanol) is the favored reaction overthe reverse reaction (i.e. the conversion of isobutanol toisobutyraldehyde).

The strategies described above generally lead to a decrease inisobutyrate yield, which is accompanied by an increase in isobutanolyield. Hence, the above strategies are useful for decreasing theisobutyrate yield and/or titer and for increasing the ratio ofisobutanol yield over isobutyrate yield.

In one embodiment, the isobutyrate yield (mol isobutyrate per molglucose) is less than about 5%. In another embodiment, the isobutyrateyield (mol isobutyrate per mol glucose) is less than about 1%. In yetanother embodiment, the isobutyrate yield (mol isobutyrate per molglucose) is less than about 0.5%, less than about 0.1%, less than about0.05%, or less than about 0.01%.

In one embodiment, the isobutanol to isobutyrate yield ratio is at leastabout 2. In another embodiment, the isobutanol to isobutyrate yield isat least about 5. In yet another embodiment, the isobutanol toisobutyrate yield ratio at least about 20, at least about 100, at leastabout 500, or at least about 1000.

The recombinant microorganisms described herein which produce abeneficial metabolite derived from a biosynthetic pathway which uses analdehyde as an intermediate may be further engineered to reduce oreliminate enzymatic activity for the conversion of pyruvate to productsother than a 3-keto acid (e.g., acetolactate and/or2-aceto-2-hydroxybutyrate). In one embodiment, the enzymatic activity ofpyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvateoxidase, pyruvate dehydrogenase, and/or glycerol-3-phosphatedehydrogenase (GPD) is reduced or eliminated.

In a specific embodiment, the beneficial metabolite is produced in arecombinant PDC-minus GPD-minus yeast microorganism that overexpressesan acetolactate synthase (ALS) gene. In another specific embodiment, theALS is encoded by the B. subtilis alsS.

Reduced Accumulation of 3-Hydroxyacid by-Products and Acid by-Products

The present inventors describe herein multiple strategies for reducingthe conversion of a 3-keto acid intermediate to a corresponding3-hydroxyacid by-product, a process which is accompanied by an increasein the yield of desirable metabolites. The present inventors alsodescribe herein multiple strategies for reducing the conversion of analdehyde intermediate to a corresponding acid by-product, a processwhich is accompanied by a further increase in the yield of desirablemetabolites.

Accordingly, in one aspect, the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses a 3-keto acidas an intermediate and an aldehyde as an intermediate, wherein saidrecombinant microorganism is (i) substantially free of an enzyme thatcatalyzes the conversion of the 3-keto acid intermediate to a3-hydroxyacid by-product and (ii) substantially free of an enzyme thatcatalyzes the conversion of an aldehyde to an acid by-product. In oneembodiment, the 3-keto acid intermediate is acetolactate. Thebiosynthetic pathway which uses acetolactate and an aldehyde asintermediates may be selected from a pathway for the biosynthesis ofisobutanol, 1-butanol, and 3-methyl-1-butanol. In another embodiment,the 3-keto acid intermediate is 2-aceto-2-hydroxybutyrate. Thebiosynthetic pathway which uses 2-aceto-2-hydroxybutyrate and analdehyde as intermediates may be selected from a pathway for thebiosynthesis of 2-methyl-1-butanol, 3-methyl-1-pentanol,4-methyl-1-hexanol, and 5-methyl-1-heptanol.

In another aspect, the invention is directed to a recombinantmicroorganism comprising a biosynthetic pathway which uses a 3-keto acidas an intermediate and an aldehyde as an intermediate, wherein saidrecombinant microorganism is (i) engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion of the3-keto acid intermediate to a 3-hydroxyacid by-product and (ii)engineered to reduce or eliminate the expression or activity of one ormore enzymes catalyzing the conversion of the aldehyde to an acidby-product. In one embodiment, the 3-keto acid intermediate isacetolactate. The biosynthetic pathway which uses acetolactate and analdehyde as intermediates may be selected from a pathway for thebiosynthesis of isobutanol, 1-butanol, and 3-methyl-1-butanol. Inanother embodiment, the 3-keto acid intermediate is2-aceto-2-hydroxybutyrate. The biosynthetic pathway which uses2-aceto-2-hydroxybutyrate and an aldehyde as intermediates may beselected from a pathway for the biosynthesis of 2-methyl-1-butanol,3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.

In various embodiments described herein, the protein involved incatalyzing the conversion of the 3-keto acid intermediate to the3-hydroxyacid by-product is a ketoreductase. In an exemplary embodiment,the ketoreductase is a 3-ketoacid reductase (3-KAR). In a furtherexemplary embodiment, the 3-ketoacid reductase is the S. cerevisiaeYMR226C (SEQ ID NO: 1) protein or a homolog or variant thereof. In oneembodiment, the homolog may be selected from the group consisting ofVanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomyces castellii (SEQID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomyces bayanus (SEQID NO: 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), K. lactis (SEQ IDNO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyces kluyveri (SEQ IDNO: 9), Kluyveromyces thermotolerans (SEQ ID NO: 10), Kluyveromyceswaltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12), Debaromyceshansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14), Candidadubliniensis (SEQ ID NO: 15), Candida albicans (SEQ ID NO: 16), Yarrowialipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO: 18),Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO: 20),Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQ ID NO:22), and Kluyveromyces marxianus (SEQ ID NO: 23).

In various embodiments described herein, the protein involved incatalyzing the conversion of an aldehyde to an acid by-product is analdehyde dehydrogenase (ALDH). In one embodiment, the aldehydedehydrogenase is encoded by a gene selected from the group consisting ofALD2, ALD3, ALD4, ALD5, ALD6, and HFD1, and homologs and variantsthereof. In an exemplary embodiment, the aldehyde dehydrogenase is theS. cerevisiae aldehyde dehydrogenase ALD6 (SEQ ID NO: 25) or homolog orvariant thereof. In one embodiment, the homolog may be selected from thegroup consisting of Saccharomyces castelli (SEQ ID NO: 26), Candidaglabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO: 28),Kluyveromyces lactis (SEQ ID NO: 29), Kluyveromyces thermotolerans (SEQID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomycescerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQID NO: 33), Saccharomyces cerevisiae EC1118 (SEQ ID NO: 34),Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomycescerevisiae AWR11631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a(SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38), Kluyveromycesmarxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40),and Schizosaccharomyces pombe (SEQ ID NO: 41).

The recombinant microorganisms described herein which produce abeneficial metabolite derived from a biosynthetic pathway which uses a3-keto acid and an aldehyde as an intermediate may be further engineeredto reduce or eliminate enzymatic activity for the conversion of pyruvateto products other than a 3-keto acid (e.g., acetolactate and/or2-aceto-2-hydroxybutyrate). In one embodiment, the enzymatic activity ofpyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvateoxidase, pyruvate dehydrogenase, and/or glycerol-3-phosphatedehydrogenase (GPD) is reduced or eliminated.

In a specific embodiment, the beneficial metabolite is produced in arecombinant PDC-minus GPD-minus yeast microorganism that overexpressesan acetolactate synthase (ALS) gene. In another specific embodiment, theALS is encoded by the B. subtilis alsS.

Illustrative Embodiments of Strategies for Reducing Accumulation of3-Hydroxyacid By-Products and/or Acid by-Products

In a specific illustrative embodiment, the recombinant microorganismcomprises an isobutanol producing metabolic pathway of whichacetolactate and isobutyraldehyde are intermediates, wherein saidrecombinant microorganism is substantially free of enzymes catalyzingthe conversion of the acetolactate intermediate to DH2MB and of theisobutyraldehyde intermediate to isobutyrate. In another specificembodiment, the recombinant microorganism comprises an isobutanolproducing metabolic pathway of which acetolactate and isobutyraldehydeare intermediates, wherein said recombinant microorganism is (i)engineered to reduce or eliminate the expression or activity of one ormore enzymes catalyzing the conversion of acetolactate to DH2MB and (ii)engineered to reduce or eliminate the expression or activity of one ormore enzymes catalyzing the conversion of isobutyraldehyde toisobutyrate. In one embodiment, the enzyme catalyzing the conversion ofacetolactate to DH2MB is a 3-ketoacid reductase (3-KAR). In anotherembodiment, the enzyme catalyzing the conversion of isobutyraldehyde toisobutyrate is an aldehyde dehydrogenase (ALDH). A non-limiting exampleof such a pathway in which a 3-ketoacid reductase (3-KAR) and analdehyde dehydrogenase (ALDH) are eliminated is depicted in FIG. 5.

In a further specific illustrative embodiment, the recombinantmicroorganism comprises a 3-methyl-1-butanol producing metabolic pathwayof which acetolactate and 3-methyl-1-butanal are intermediates, whereinsaid recombinant microorganism is substantially free of enzymescatalyzing the conversion of the acetolactate intermediate to DH2MB andof the 3-methyl-1-butanal intermediate to 3-methyl-1-butyrate. Inanother specific embodiment, the recombinant microorganism comprises a3-methyl-1-butanol producing metabolic pathway of which acetolactate and3-methyl-1-butanal are intermediates, wherein said recombinantmicroorganism is (i) engineered to reduce or eliminate the expression oractivity of one or more enzymes catalyzing the conversion ofacetolactate to DH2MB and (ii) engineered to reduce or eliminate theexpression or activity of one or more enzymes catalyzing the conversionof 3-methyl-1-butanal to 3-methyl-1-butyrate. In one embodiment, theenzyme catalyzing the conversion of acetolactate to DH2MB is a3-ketoacid reductase (3-KAR). In another embodiment, the enzymecatalyzing the conversion of 3-methyl-1-butanal to 3-methyl-1-butyrateis an aldehyde dehydrogenase (ALDH). A non-limiting example of such apathway in which a 3-ketoacid reductase (3-KAR) and an aldehydedehydrogenase (ALDH) are eliminated is depicted in FIG. 6.

In a further specific illustrative embodiment, the recombinantmicroorganism comprises a 2-methyl-1-butanol producing metabolic pathwayof which acetolactate and 2-methyl-1-butanal are intermediates, whereinsaid recombinant microorganism is substantially free of enzymescatalyzing the conversion of the 2-aceto-2-hydroxybutyrate intermediateto 2-ethyl-2,3-dihydroxybutyrate and of the 2-methyl-1-butanalintermediate to 2-methyl-1-butyrate. In another specific embodiment, therecombinant microorganism comprises a 2-methyl-1-butanol producingmetabolic pathway of which 2-aceto-2-hydroxybutyrate and2-methyl-1-butanal are intermediates, wherein said recombinantmicroorganism is (i) engineered to reduce or eliminate the expression oractivity of one or more enzymes catalyzing the conversion of2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrate and (ii)engineered to reduce or eliminate the expression or activity of one ormore enzymes catalyzing the conversion of 2-methyl-1-butanal to2-methyl-1-butyrate. In one embodiment, the enzyme catalyzing theconversion of 2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrateis a 3-ketoacid reductase (3-KAR). In another embodiment, the enzymecatalyzing the conversion of 2-methyl-1-butanal to 2-methyl-1-butyrateis an aldehyde dehydrogenase (ALDH). A non-limiting example of such apathway in which a 3-ketoacid reductase (3-KAR) and an aldehydedehydrogenase (ALDH) are eliminated is depicted in FIG. 7.

Overexpression of Enzymes Converting DH2MB into Isobutanol PathwayIntermediates

A different approach to reduce or eliminate the production of2,3-dihydroxy-2-methylbutanoic acid (CAS#14868-24-7) in isobutanolproducing yeast is to overexpress an enzyme that converts DH2MB into anisobutanol pathway intermediate. One way to accomplish this is throughthe use of an enzyme that catalyzes the interconversion of DH2MB andacetolactate, but favors the oxidation of DH2MB. Therefore, in oneembodiment, the present invention provides a recombinant microorganismfor producing isobutanol, wherein said recombinant microorganismoverexpresses an endogenous or heterologous protein capable ofconverting DH2MB into acetolactate.

In one embodiment, the endogenous or heterologous protein kineticallyfavors the oxidative reaction. In another embodiment, the endogenous orheterologous protein has a low K_(M) for DH2MB and a high K_(M) foracetolactate. In yet another embodiment, the endogenous or heterologousprotein has a low K_(M) for the oxidized form of its cofactor and a highK_(M) for the corresponding reduced form of its cofactor. In yet anotherembodiment, the endogenous or heterologous protein has a higher k_(cat)for the oxidative reaction than for the reductive direction. Thisendogenous or heterologous protein should preferably have the ability touse a redox cofactor with a high concentration of its oxidized formversus its reduced form.

In one embodiment, the endogenous or heterologous protein is encoded bya gene selected from the group consisting of YAL060W, YJR159W, YGL157W,YBL114W, YOR120W, YKL055C, YBR159W, YBR149W, YDL168W, YDR368W, YLR426W,YCR107W, YILL24W, YML054C, YOL151W, YMR318C, YBR046C, YHR104W, YIR036C,YDL174C, YDR541C, YBR145W, YGL039W, YCR105W, YDL124W, YIR035C, YFL056C,YNL274c, YLR255C, YGL185C, YGL256W, YJR096W, YJR155W, YPL275W, YOR388C,YLR070C, YMR083W, YER081W, YJR139C, YDL243C, YPL113C, YOL165C, YML086C,YMR303C, YDL246C, YLR070C, YHR063C, YNL331C, YFL057C, YIL155C, YOL086C,YAL061W, YDR127W, YPR127W, YCL018W, YIL074C, YIL124W, and YEL071W. Inaddition, heterologous genes can be overexpressed in isobutanolproducing yeast. For examples beta-hydroxy acid dehydrogenases(EC1.1.1.45 and EC1.1.1.60) would be candidates for overexpression.

In another embodiment, the endogenous or heterologous proteinkinetically that favors the reductive reaction is engineered to favorthe oxidative reaction. In another embodiment, the protein is engineeredto have a low K_(M) for DH2MB and a high K_(M) for acetolactate. In yetanother embodiment, the protein is engineered to have a low K_(M) forthe oxidized form of its cofactor and a high K_(M) for the correspondingreduced form of its cofactor. In yet another embodiment, the protein isengineered to have a higher k_(cat) for the oxidative reaction than forthe reductive direction. This engineered protein should preferably havethe ability to use a redox cofactor with a high concentration of itsoxidized form versus its reduced form.

Alternatively, an enzyme could be overexpressed that isomerizes DH2MBinto DHIV. This approach represents a novel pathway for the productionof isobutanol from pyruvate. Thus, in one embodiment, the presentinvention provides a recombinant microorganism for producing isobutanol,wherein said recombinant microorganism overexpresses an endogenous orheterologous protein capable of converting DH2MB into2,3-dihydroxyisovalerate.

Overexpression of Enzymes Converting 2-Ethyl-2,3-Dihydroxybutanoate intoBiosynthetic Pathway Intermediates

A different approach to reduce or eliminate the production of2-ethyl-2,3-dihydroxybutanoate in yeast is to overexpress an enzyme thatconverts 2-ethyl-2,3-dihydroxybutanoate into a biosynthetic pathwayintermediate. This approach is useful for any biosynthetic pathway whichuses 2-aceto-2-hydroxybutyrate as an intermediate, including, but notlimited to, 2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol,4-methyl-1-hexanol, and 5-methyl-1-heptanol. One way to accomplish thisis through the use of an enzyme that catalyzes the interconversion of2-ethyl-2,3-dihydroxybutanoate and 2-aceto-2-hydroxybutyrate, but favorsthe oxidation of 2-ethyl-2,3-dihydroxybutanoate. Therefore, in oneembodiment, the present invention provides a recombinant microorganismfor producing a product selected from 2-methyl-1-butanol, isoleucine,3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol whereinsaid recombinant microorganism overexpresses an endogenous orheterologous protein capable of converting2-ethyl-2,3-dihydroxybutanoate into 2-aceto-2-hydroxybutyrate.

In one embodiment, the endogenous or heterologous protein kineticallyfavors the oxidative reaction. In another embodiment, the endogenous orheterologous protein has a low K_(M) for 2-ethyl-2,3-dihydroxybutanoateand a high K_(M) for 2-aceto-2-hydroxybutyrate. In yet anotherembodiment, the endogenous or heterologous protein has a low K_(M) forthe oxidized form of its cofactor and a high K_(M) for the correspondingreduced form of its cofactor. In yet another embodiment, the endogenousor heterologous protein has a higher k_(cat) for the oxidative reactionthan for the reductive direction. This endogenous or heterologousprotein should preferably have the ability to use a redox cofactor witha high concentration of its oxidized form versus its reduced form.

In one embodiment, the endogenous or heterologous protein is encoded bya gene selected from the group consisting of YAL060W, YJR159W, YGL157W,YBL114W, YOR120W, YKL055C, YBR159W, YBR149W, YDL168W, YDR368W, YLR426W,YCR107W, YILL24W, YML054C, YOL151W, YMR318C, YBR046C, YHR104W, YIR036C,YDL174C, YDR541C, YBR145W, YGL039W, YCR105W, YDL124W, YIR035C, YFL056C,YNL274c, YLR255C, YGL185C, YGL256W, YJR096W, YJR155W, YPL275W, YOR388C,YLR070C, YMR083W, YER081W, YJR139C, YDL243C, YPL113C, YOL165C, YML086C,YMR303C, YDL246C, YLR070C, YHR063C, YNL331C, YFL057C, YIL155C, YOL086C,YAL061W, YDR127W, YPR127W, YCL018W, YIL074C, YIL124W, and YEL071W. Inaddition, heterologous genes can be overexpressed in isoleucineproducing yeast. For examples beta-hydroxy acid dehydrogenases(EC1.1.1.45 and EC1.1.1.60) would be candidates for overexpression.

Alternatively an enzyme could be overexpressed that isomerizes2-ethyl-2,3-dihydroxybutanoate into 2,3-dihydroxy-3-methylvalerate. Thisapproach represents a novel pathway for the production of2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol,and 5-methyl-1-heptanol from pyruvate. Thus, in one embodiment, thepresent invention provides a recombinant microorganism for producing aproduct selected from 2-methyl-1-butanol, isoleucine,3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol,wherein said recombinant microorganism overexpresses an endogenous orheterologous protein capable of converting2-ethyl-2,3-dihydroxybutanoate into α,β-dihydroxy-β-methylvalerate.

Use of Overexpressed Ketol-Acid Reductoisomerase (KARI) and/or ModifiedKetol-Acid Reductoisomerase (KARI) to Reduce the Production of DH2MB

As described herein, the conversion of acetolactate to DH2MB competeswith the isobutanol pathway for the intermediate acetolactate. In thecurrent yeast isobutanol production strains, ketol-acid reductoisomerase(KARI) catalyzes the conversion of acetolactate to DHIV.

In one embodiment, the present invention provides recombinantmicroorganisms having an overexpressed ketol-acid reductoisomerase(KARI), which catalyzes the conversion of acetolactate to2,3-dihydroxyisovalerate (DHIV). The overexpression of KARI has theeffect of reducing DH2MB production. In one embodiment, the KARI has atleast 0.01 U/mg of activity in the lysate. In another embodiment, theKARI has at least 0.03 U/mg of activity in the lysate. In yet anotherembodiment, the KARI has at least 0.05, 0.1, 0.5, 1, 2, 5, or 10 U/mg ofactivity in the lysate.

In a preferred embodiment, the overexpressed KARI is engineered toexhibit a reduced K_(M) for acetolactate as compared to a wild-type orparental KARI. The use of the modified KARI with lower K_(M) foracetolactate is expected to reduce the production of the by-productDH2MB. A KARI with lower substrate K_(M) is identified by screeninghomologs. In the alternative, the KARI can be engineered to exhibitreduced K_(M) by directed evolution using techniques known in the art.

In each of these embodiments, the KARI may be a variant enzyme thatutilizes NADH (rather than NADPH) as a co-factor. Such enzymes aredescribed in the commonly owned and co-pending publication, US2010/0143997, which is herein incorporated by reference in its entiretyfor all purposes.

Use of Overexpressed Dihydroxy Acid Dehydratase (DHAD) to Reduce theProduction of DH2MB

As described herein, the present inventors have found thatoverexpression of the isobutanol pathway enzyme, dihydroxyaciddehydratase (DHAD), reduces the production of the by-product, DH2MB.

Accordingly, in one embodiment, the present invention providesrecombinant microorganisms having an dihydroxyacid dehydratase (DHAD),which catalyzes the conversion of 2,3-dihydroxyisovalerate (DHIV) to2-ketoisovalerate (KIV). The overexpression of DHAD has the effect ofreducing DH2MB production. In one embodiment, the DHAD has at least 0.01U/mg of activity in the lysate. In another embodiment, the DHAD has atleast 0.03 U/mg of activity in the lysate. In yet another embodiment,the DHAD has at least 0.05, 0.1, 0.5, 1, 2, 5, or 10 U/mg of activity inthe lysate.

Recombinant Microorganisms for the Production of 3-Hydroxyacids

The present invention provides in additional aspects recombinantmicroorganisms for the production of 3-hydroxyacids as a product or ametabolic intermediate. In one embodiment, these 3-hydroxyacid-producingrecombinant microorganisms express acetolactate synthase (ALS) and a3-ketoacid reductase catalyzing the reduction of 2-acetolactate toDH2MB. In another embodiment, these 3-hydroxyacid-producing recombinantmicroorganisms express acetolactate synthase (ALS) and a 3-ketoacidreductase catalyzing the reduction of 2-aceto-2-hydroxybutyrate into2-ethyl-2,3-dihydroxybutyrate.

These 3-hydroxyacid-producing recombinant microorganisms may be furtherengineered to reduce or eliminate enzymatic activity for the conversionof pyruvate to products other than acetolactate. In one embodiment, theenzymatic activity of pyruvate decarboxylase (PDC), lactatedehydrogenase (LDH), pyruvate oxidase, pyruvate dehydrogenase, and/orglycerol-3-phosphate dehydrogenase (GPD) is reduced or eliminated.

In a specific embodiment, DH2MB is produced in a recombinant PDC-minusGPD-minus yeast microorganism that overexpresses an ALS gene andexpresses a 3-ketoacid reductase. In one embodiment, the 3-ketoacidreductase is natively expressed. In another embodiment, the 3-ketoacidreductase is heterologously expressed. In yet another embodiment, the3-ketoacid reductase is overexpressed. In a specific embodiment, the3-ketoacid reductase is encoded by the S. cerevisiae TMA29 gene or ahomolog thereof. In another specific embodiment, the ALS is encoded bythe B. subtilis AlsS.

In another specific embodiment, 2-ethyl-2,3-dihydroxybutyrate isproduced in a recombinant PDC-minus GPD-minus yeast microorganism thatoverexpresses an ALS gene and expresses a 3-ketoacid reductase. In oneembodiment, the 3-ketoacid reductase is natively expressed. In anotherembodiment, the 3-ketoacid reductase is heterologously expressed. In yetanother embodiment, the 3-ketoacid reductase is overexpressed. In aspecific embodiment, the 3-ketoacid reductase is encoded by the S.cerevisiae TMA29 gene or a homolog thereof. In another specificembodiment, the ALS is encoded by the B. subtilis AlsS.

In accordance with these additional aspects, the present invention alsoprovides a method of producing 2,3-dihydroxy-2-methylbutanoic acid(DH2MB), comprising: (a) providing a DH2MB-producing recombinantmicroorganism that expresses acetolactate synthase (ALS) and a3-ketoacid reductase catalyzing the reduction of 2-acetolactate toDH2MB, and (b) cultivating said recombinant microorganism in a culturemedium containing a feedstock providing the carbon source, until arecoverable quantity of DH2MB is produced.

In accordance with these additional aspects, the present invention alsoprovides a method of producing 2-ethyl-2,3-dihydroxybutyrate,comprising: (a) providing a 2-ethyl-2,3-dihydroxybutyrate-producingrecombinant microorganism that expresses acetolactate synthase (ALS) anda 3-ketoacid reductase catalyzing the reduction of2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrate, and (b)cultivating said recombinant microorganism in a culture mediumcontaining a feedstock providing the carbon source, until a recoverablequantity of 2-ethyl-2,3-dihydroxybutyrate is produced.

Recombinant Microorganisms for the Production of Acid Products

The present invention provides in additional aspects recombinantmicroorganisms for the production of acid products derived fromaldehydes. In one embodiment, these acid product producing recombinantmicroorganisms express an aldehyde dehydrogenase catalyzing theconversion of an aldehyde to a corresponding acid product. These acidproduct producing recombinant microorganisms may be further engineeredto reduce or eliminate competing enzymatic activity for the undesirableconversion of metabolites upstream of the desired acid product.

In a specific embodiment, the acid product is produced in a recombinantyeast microorganism that overexpresses an aldehyde dehydrogenase. In oneembodiment, the aldehyde dehydrogenase is natively expressed. In anotherembodiment, the aldehyde dehydrogenase is heterologously expressed. Inyet another embodiment, the aldehyde dehydrogenase is overexpressed. Ina specific embodiment, the aldehyde dehydrogenase is encoded by the S.cerevisiae ALD6 gene or a homolog thereof.

In accordance with this additional aspect, the present invention alsoprovides a method of producing an acid product, comprising: (a)providing an acid product-producing recombinant microorganism thatexpresses an aldehyde dehydrogenase catalyzing the conversion of analdehyde to acid product, and (b) cultivating said recombinantmicroorganism in a culture medium containing a feedstock providing thecarbon source, until a recoverable quantity of the desired acid productis produced.

The Microorganism in General

The recombinant microorganisms provided herein can express a pluralityof heterologous and/or native enzymes involved in pathways for theproduction of beneficial metabolites such as isobutanol, 2-butanol,1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl, valine,leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol, coenzyme A,2-methyl-1-butanol, isoleucine, 1-pentanol, 1-hexanol,3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol,5-methyl-1-heptanol, and 1-propanol from a suitable carbon source. Anon-limiting list of beneficial metabolites produced in engineeredbiosynthetic pathways is found herein at Tables 1-3.

As described herein, “engineered” or “modified” microorganisms areproduced via the introduction of genetic material into a host orparental microorganism of choice and/or by modification of theexpression of native genes, thereby modifying or altering the cellularphysiology and biochemistry of the microorganism. Through theintroduction of genetic material and/or the modification of theexpression of native genes the parental microorganism acquires newproperties, e.g., the ability to produce a new, or greater quantitiesof, an intracellular and/or extracellular metabolite. As describedherein, the introduction of genetic material into and/or themodification of the expression of native genes in a parentalmicroorganism results in a new or modified ability to produce beneficialmetabolites such as isobutanol, 2-butanol, 1-butanol, 2-butanone,2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid,isobutylene, 3-methyl-1-butanol, coenzyme A, 2-methyl-1-butanol,isoleucine, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol,4-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, and1-propanol from a suitable carbon source. The genetic materialintroduced into and/or the genes modified for expression in the parentalmicroorganism contains gene(s), or parts of genes, coding for one ormore of the enzymes involved in a biosynthetic pathway for theproduction of one or more metabolites selected from isobutanol,2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl,valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol,coenzyme A, 2-methyl-1-butanol, isoleucine, 1-pentanol, 1-hexanol,3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol,5-methyl-1-heptanol, and 1-propanol and may also include additionalelements for the expression and/or regulation of expression of thesegenes, e.g., promoter sequences.

In addition to the introduction of a genetic material into a host orparental microorganism, an engineered or modified microorganism can alsoinclude alteration, disruption, deletion or knocking-out of a gene orpolynucleotide to alter the cellular physiology and biochemistry of themicroorganism. Through the alteration, disruption, deletion orknocking-out of a gene or polynucleotide the microorganism acquires newor improved properties (e.g., the ability to produce a new metabolite orgreater quantities of an intracellular metabolite, to improve the fluxof a metabolite down a desired pathway, and/or to reduce the productionof by-products).

Recombinant microorganisms provided herein may also produce metabolitesin quantities not available in the parental microorganism. A“metabolite” refers to any substance produced by metabolism or asubstance necessary for or taking part in a particular metabolicprocess. A metabolite can be an organic compound that is a startingmaterial (e.g., glucose or pyruvate), an intermediate (e.g.,2-ketoisovalerate), or an end product (e.g., isobutanol) of metabolism.Metabolites can be used to construct more complex molecules, or they canbe broken down into simpler ones. Intermediate metabolites may besynthesized from other metabolites, perhaps used to make more complexsubstances, or broken down into simpler compounds, often with therelease of chemical energy.

The disclosure identifies specific genes useful in the methods,compositions and organisms of the disclosure; however it will berecognized that absolute identity to such genes is not necessary. Forexample, changes in a particular gene or polynucleotide comprising asequence encoding a polypeptide or enzyme can be performed and screenedfor activity. Typically such changes comprise conservative mutations andsilent mutations. Such modified or mutated polynucleotides andpolypeptides can be screened for expression of a functional enzyme usingmethods known in the art.

Due to the inherent degeneracy of the genetic code, otherpolynucleotides which encode substantially the same or functionallyequivalent polypeptides can also be used to clone and express thepolynucleotides encoding such enzymes.

As will be understood by those of skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms typically use a subset of these codons. The codonsthat are utilized most often in a species are called optimal codons, andthose not utilized very often are classified as rare or low-usagecodons. Codons can be substituted to reflect the preferred codon usageof the host, in a process sometimes called “codon optimization” or“controlling for species codon bias.”

Optimized coding sequences containing codons preferred by a particularprokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res. 17:477-508) can be prepared, for example, to increase the rate oftranslation or to produce recombinant RNA transcripts having desirableproperties, such as a longer half-life, as compared with transcriptsproduced from a non-optimized sequence. Translation stop codons can alsobe modified to reflect host preference. For example, typical stop codonsfor S. cerevisiae and mammals are UAA and UGA, respectively. The typicalstop codon for monocotyledonous plants is UGA, whereas insects and E.coli commonly use UAA as the stop codon (Dalphin et al., 1996, NuclAcids Res. 24: 216-8). Methodology for optimizing a nucleotide sequencefor expression in a plant is provided, for example, in U.S. Pat. No.6,015,891, and the references cited therein.

Those of skill in the art will recognize that, due to the degeneratenature of the genetic code, a variety of DNA compounds differing intheir nucleotide sequences can be used to encode a given enzyme of thedisclosure. The native DNA sequence encoding the biosynthetic enzymesdescribed above are referenced herein merely to illustrate an embodimentof the disclosure, and the disclosure includes DNA compounds of anysequence that encode the amino acid sequences of the polypeptides andproteins of the enzymes utilized in the methods of the disclosure. Insimilar fashion, a polypeptide can typically tolerate one or more aminoacid substitutions, deletions, and insertions in its amino acid sequencewithout loss or significant loss of a desired activity. The disclosureincludes such polypeptides with different amino acid sequences than thespecific proteins described herein so long as the modified or variantpolypeptides have the enzymatic anabolic or catabolic activity of thereference polypeptide. Furthermore, the amino acid sequences encoded bythe DNA sequences shown herein merely illustrate embodiments of thedisclosure.

In addition, homologs of enzymes useful for generating metabolites areencompassed by the microorganisms and methods provided herein.

As used herein, two proteins (or a region of the proteins) aresubstantially homologous when the amino acid sequences have at leastabout 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percentidentity of two amino acid sequences, or of two nucleic acid sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). In one embodiment, thelength of a reference sequence aligned for comparison purposes is atleast 30%, typically at least 40%, more typically at least 50%, evenmore typically at least 60%, and even more typically at least 70%, 80%,90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

When “homologous” is used in reference to proteins or peptides, it isrecognized that residue positions that are not identical often differ byconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of homology may be adjusted upwardsto correct for the conservative nature of the substitution. Means formaking this adjustment are well known to those of skill in the art (See,e.g., Pearson W. R., 1994, Methods in Mol Biol 25: 365-89).

The following six groups each contain amino acids that are conservativesubstitutions for one another: 1) Serine (S), Threonine (T); 2) AsparticAcid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A),Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Sequence homology for polypeptides, which is also referred to as percentsequence identity, is typically measured using sequence analysissoftware. See commonly owned and co-pending application US 2009/0226991.A typical algorithm used comparing a molecule sequence to a databasecontaining a large number of sequences from different organisms is thecomputer program BLAST. When searching a database containing sequencesfrom a large number of different organisms, it is typical to compareamino acid sequences. Database searching using amino acid sequences canbe measured by algorithms described in commonly owned and co-pendingapplication US 2009/0226991.

It is understood that a range of microorganisms can be modified toinclude a recombinant metabolic pathway suitable for the production ofbeneficial metabolites from acetolactate- and/or aldehydeintermediate-requiring biosynthetic pathways. In various embodiments,microorganisms may be selected from yeast microorganisms. Yeastmicroorganisms for the production of a metabolite such as isobutanol,2-butanol, 1-butanol, 2-butanone, 2,3-butanediol, acetoin, diacetyl,valine, leucine, pantothenic acid, isobutylene, 3-methyl-1-butanol,coenzyme A, 2-methyl-1-butanol, isoleucine, 1-pentanol, 1-hexanol,3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol,5-methyl-1-heptanol, and 1-propanol may be selected based on certaincharacteristics:

One characteristic may include the property that the microorganism isselected to convert various carbon sources into beneficial metabolitessuch as isobutanol, 2-butanol, 1-butanol, 2-butanone, 2,3-butanediol,acetoin, diacetyl, valine, leucine, pantothenic acid, isobutylene,3-methyl-1-butanol, coenzyme A, 2-methyl-1-butanol, isoleucine,1-pentanol, 1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol,4-methyl-1-hexanol, 5-methyl-1-heptanol, and 1-propanol. The term“carbon source” generally refers to a substance suitable to be used as asource of carbon for prokaryotic or eukaryotic cell growth. Examples ofsuitable carbon sources are described in commonly owned and co-pendingapplication US 2009/0226991. Accordingly, in one embodiment, therecombinant microorganism herein disclosed can convert a variety ofcarbon sources to products, including but not limited to glucose,galactose, mannose, xylose, arabinose, lactose, sucrose, and mixturesthereof.

The recombinant microorganism may thus further include a pathway for theproduction of isobutanol, 2-butanol, 1-butanol, 2-butanone,2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid,isobutylene, 3-methyl-1-butanol, coenzyme A, 2-methyl-1-butanol,isoleucine, 1-pentanol, 1-hexanol, 3-methyl-1-pentanol,4-methyl-1-pentanol, 4-methyl-1-hexanol, 5-methyl-1-heptanol, and1-propanol from five-carbon (pentose) sugars including xylose. Mostyeast species metabolize xylose via a complex route, in which xylose isfirst reduced to xylitol via a xylose reductase (XR) enzyme. The xylitolis then oxidized to xylulose via a xylitol dehydrogenase (XDH) enzyme.The xylulose is then phosphorylated via a xylulokinase (XK) enzyme. Thispathway operates inefficiently in yeast species because it introduces aredox imbalance in the cell. The xylose-to-xylitol step uses NADH as acofactor, whereas the xylitol-to-xylulose step uses NADPH as a cofactor.Other processes must operate to restore the redox imbalance within thecell. This often means that the organism cannot grow anaerobically onxylose or other pentose sugars. Accordingly, a yeast species that canefficiently ferment xylose and other pentose sugars into a desiredfermentation product is therefore very desirable.

Thus, in one aspect, the recombinant microorganism is engineered toexpress a functional exogenous xylose isomerase. Exogenous xyloseisomerases functional in yeast are known in the art. See, e.g.,Rajgarhia et al., US2006/0234364, which is herein incorporated byreference in its entirety. In an embodiment according to this aspect,the exogenous xylose isomerase gene is operatively linked to promoterand terminator sequences that are functional in the yeast cell. In apreferred embodiment, the recombinant microorganism further has adeletion or disruption of a native gene that encodes for an enzyme(e.g., XR and/or XDH) that catalyzes the conversion of xylose toxylitol. In a further preferred embodiment, the recombinantmicroorganism also contains a functional, exogenous xylulokinase (XK)gene operatively linked to promoter and terminator sequences that arefunctional in the yeast cell. In one embodiment, the xylulokinase (XK)gene is overexpressed.

In one embodiment, the microorganism has reduced or no pyruvatedecarboxylase (PDC) activity. PDC catalyzes the decarboxylation ofpyruvate to acetaldehyde, which is then reduced to ethanol by ADH via anoxidation of NADH to NAD+. Ethanol production is the main pathway tooxidize the NADH from glycolysis. Deletion of this pathway increases thepyruvate and the reducing equivalents (NADH) available for thebiosynthetic pathway. Accordingly, deletion of PDC genes can furtherincrease the yield of desired metabolites.

In another embodiment, the microorganism has reduced or noglycerol-3-phosphate dehydrogenase (GPD) activity. GPD catalyzes thereduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate(G3P) via the oxidation of NADH to NAD+. Glycerol is then produced fromG3P by Glycerol-3-phosphatase (GPP). Glycerol production is a secondarypathway to oxidize excess NADH from glycolysis. Reduction or eliminationof this pathway would increase the pyruvate and reducing equivalents(NADH) available for the biosynthetic pathway. Thus, deletion of GPDgenes can further increase the yield of desired metabolites.

In yet another embodiment, the microorganism has reduced or no PDCactivity and reduced or no GPD activity. PDC-minus/GPD-minus yeastproduction strains are described in commonly owned and co-pendingpublications, US 2009/0226991 and US 2011/0020889, both of which areherein incorporated by reference in their entireties for all purposes.

In one embodiment, the yeast microorganisms may be selected from the“Saccharomyces Yeast Clade”, as described in commonly owned andco-pending application US 2009/0226991.

The term “Saccharomyces sensu stricto” taxonomy group is a cluster ofyeast species that are highly related to S. cerevisiae (Rainieri et al.,2003, J. Biosci Bioengin 96: 1-9). Saccharomyces sensu stricto yeastspecies include but are not limited to S. cerevisiae, S. kudriavzevii,S. mikatae, S. bayanus, S. uvarum, S. carocanis and hybrids derived fromthese species (Masneuf et al., 1998, Yeast 7: 61-72).

An ancient whole genome duplication (WGD) event occurred during theevolution of the hemiascomycete yeast and was discovered usingcomparative genomic tools (Kellis et al., 2004, Nature 428: 617-24;Dujon et al., 2004, Nature 430:35-44; Langkjaer et al., 2003, Nature428: 848-52; Wolfe et al., 1997, Nature 387: 708-13). Using this majorevolutionary event, yeast can be divided into species that diverged froma common ancestor following the WGD event (termed “post-WGD yeast”herein) and species that diverged from the yeast lineage prior to theWGD event (termed “pre-WGD yeast” herein).

Accordingly, in one embodiment, the yeast microorganism may be selectedfrom a post-WGD yeast genus, including but not limited to Saccharomycesand Candida. The favored post-WGD yeast species include: S. cerevisiae,S. uvarum, S. bayanus, S. paradoxus, S. castelli, and C. glabrata.

In another embodiment, the yeast microorganism may be selected from apre-whole genome duplication (pre-WGD) yeast genus including but notlimited to Saccharomyces, Kluyveromyces, Candida, Pichia, Issatchenkia,Debaryomyces, Hansenula, Yarrowia and, Schizosaccharomyces.Representative pre-WGD yeast species include: S. kluyveri, K.thermotolerans, K. marxianus, K. waltii, K. lactis, C. tropicalis, P.pastoris, P. anomala, P. stipitis, I. orientalis, I. occidentalis, I.scutulata, D. hansenii, H. anomala, Y. lipolytica, and S. pombe.

A yeast microorganism may be either Crabtree-negative orCrabtree-positive as described in described in commonly owned andco-pending application US 2009/0226991. In one embodiment the yeastmicroorganism may be selected from yeast with a Crabtree-negativephenotype including but not limited to the following genera:Saccharomyces, Kluyveromyces, Pichia, Issatchenkia, Hansenula, andCandida. Crabtree-negative species include but are not limited to: S.kluyveri, K. lactis, K. marxianus, P. anomala, P. stipitis, I.orientalis, I. occidentalis, I. scutulata, H. anomala, and C. utilis. Inanother embodiment, the yeast microorganism may be selected from yeastwith a Crabtree-positive phenotype, including but not limited toSaccharomyces, Kluyveromyces, Zygosaccharomyces, Debaryomyces, Pichiaand Schizosaccharomyces. Crabtree-positive yeast species include but arenot limited to: S. cerevisiae, S. uvarum, S. bayanus, S. paradoxus, S.castelli, K. thermotolerans, C. glabrata, Z. bailli, Z. rouxii, D.hansenii, P. pastorius, and S. pombe.

Another characteristic may include the property that the microorganismis that it is non-fermenting. In other words, it cannot metabolize acarbon source anaerobically while the yeast is able to metabolize acarbon source in the presence of oxygen. Nonfermenting yeast refers toboth naturally occurring yeasts as well as genetically modified yeast.During anaerobic fermentation with fermentative yeast, the main pathwayto oxidize the NADH from glycolysis is through the production ofethanol. Ethanol is produced by alcohol dehydrogenase (ADH) via thereduction of acetaldehyde, which is generated from pyruvate by pyruvatedecarboxylase (PDC). In one embodiment, a fermentative yeast can beengineered to be non-fermentative by the reduction or elimination of thenative PDC activity. Thus, most of the pyruvate produced by glycolysisis not consumed by PDC and is available for the isobutanol pathway.Deletion of this pathway increases the pyruvate and the reducingequivalents available for the biosynthetic pathway. Fermentativepathways contribute to low yield and low productivity of desiredmetabolites such as isobutanol. Accordingly, deletion of PDC genes mayincrease yield and productivity of desired metabolites such asisobutanol.

In some embodiments, the recombinant microorganisms may bemicroorganisms that are non-fermenting yeast microorganisms, including,but not limited to those, classified into a genera selected from thegroup consisting of Tricosporon, Rhodotorula, Myxozyma, or Candida. In aspecific embodiment, the non-fermenting yeast is C. xestobii.

Isobutanol-Producing Yeast Microorganisms

As described herein, in one embodiment, a yeast microorganism isengineered to convert a carbon source, such as glucose, to pyruvate byglycolysis and the pyruvate is converted to isobutanol via an isobutanolproducing metabolic pathway (See, e.g., WO/2007/050671, WO/2008/098227,and Atsumi et al., 2008, Nature 45: 86-9). Alternative pathways for theproduction of isobutanol have been described in WO/2007/050671 and inDickinson et al., 1998, J Biol Chem 273:25751-6.

Accordingly, in one embodiment, the isobutanol producing metabolicpathway to convert pyruvate to isobutanol can be comprised of thefollowing reactions:

2 pyruvate→acetolactate+CO₂  1.

acetolactate+NAD(P)H→2,3-dihydroxyisovalerate+NAD(P)⁺  2.

2,3-dihydroxyisovalerate→alpha-ketoisovalerate  3.

alpha-ketoisovalerate→isobutyraldehyde+CO₂  4.

isobutyraldehyde+NAD(P)H→isobutanol+NAD(P)⁺  5.

These reactions are carried out by the enzymes 1) Acetolactate Synthase(ALS), 2) Ketol-acid Reducto-Isomerase (KARI), 3) Dihydroxy-aciddehydratase (DHAD), 4) Keto-isovalerate decarboxylase (KIVD), and 5) anAlcohol dehydrogenase (ADH) (FIG. 1). In another embodiment, the yeastmicroorganism is engineered to overexpress these enzymes. For example,these enzymes can be encoded by native genes. Alternatively, theseenzymes can be encoded by heterologous genes. For example, ALS can beencoded by the alsS gene of B. subtilis, alsS of L. lactis, or the ilvKgene of K. pneumonia. For example, KARI can be encoded by the ilvC genesof E. coli, C. glutamicum, M. maripaludis, or Piromyces sp E2. Forexample, DHAD can be encoded by the ilvD genes of E. coli, C.glutamicum, or L. lactis. For example, KIVD can be encoded by the kivDgene of L. lactis. ADH can be encoded by ADH2, ADH6, or ADH7 of S.cerevisiae or adhA of L. lactis.

In one embodiment, pathway steps 2 and 5 may be carried out by KARI andADH enzymes that utilize NADH (rather than NADPH) as a co-factor. Suchenzymes are described in the commonly owned and co-pending publication,US 2010/0143997, which is herein incorporated by reference in itsentirety for all purposes. The present inventors have found thatutilization of NADH-dependent KARI and ADH enzymes to catalyze pathwaysteps 2 and 5, respectively, surprisingly enables production ofisobutanol under anaerobic conditions. Thus, in one embodiment, therecombinant microorganisms of the present invention may use anNADH-dependent KARI to catalyze the conversion of acetolactate (+NADH)to produce 2,3-dihydroxyisovalerate. In another embodiment, therecombinant microorganisms of the present invention may use anNADH-dependent ADH to catalyze the conversion of isobutyraldehyde(+NADH) to produce isobutanol. In yet another embodiment, therecombinant microorganisms of the present invention may use both anNADH-dependent KARI to catalyze the conversion of acetolactate (+NADH)to produce 2,3-dihydroxyisovalerate, and an NADH-dependent ADH tocatalyze the conversion of isobutyraldehyde (+NADH) to produceisobutanol.

In another embodiment, the yeast microorganism may be engineered to haveincreased ability to convert pyruvate to isobutanol. In one embodiment,the yeast microorganism may be engineered to have increased ability toconvert pyruvate to isobutyraldehyde. In another embodiment, the yeastmicroorganism may be engineered to have increased ability to convertpyruvate to keto-isovalerate. In another embodiment, the yeastmicroorganism may be engineered to have increased ability to convertpyruvate to 2,3-dihydroxyisovalerate. In another embodiment, the yeastmicroorganism may be engineered to have increased ability to convertpyruvate to acetolactate.

Furthermore, any of the genes encoding the foregoing enzymes (or anyothers mentioned herein (or any of the regulatory elements that controlor modulate expression thereof)) may be optimized by genetic/proteinengineering techniques, such as directed evolution or rationalmutagenesis, which are known to those of ordinary skill in the art. Suchaction allows those of ordinary skill in the art to optimize the enzymesfor expression and activity in yeast.

In addition, genes encoding these enzymes can be identified from otherfungal and bacterial species and can be expressed for the modulation ofthis pathway. A variety of organisms could serve as sources for theseenzymes, including, but not limited to, Saccharomyces spp., including S.cerevisiae and S. uvarum, Kluyveromyces spp., including K.thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenulaspp., including H. polymorpha, Candida spp., Trichosporon spp.,Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis,Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe,Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp.Sources of genes from anaerobic fungi include, but not limited to,Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources ofprokaryotic enzymes that are useful include, but not limited to,Escherichia. coli, Zymomonas mobilis, Staphylococcus aureus, Bacillusspp., Clostridium spp., Corynebacterium spp., Pseudomonas spp.,Lactococcus spp., Enterobacter spp., and Salmonella spp.

In one embodiment, the invention is directed to a recombinantmicroorganism for producing isobutanol, wherein said recombinantmicroorganism comprises an isobutanol producing metabolic pathway andwherein said microorganism is engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofacetolactate to DH2MB. In some embodiments, the enzyme catalyzing theconversion of acetolactate to DH2MB is a 3-ketoacid reductase (3-KAR).In a specific embodiment, the 3-ketoacid reductase is encoded by the S.cerevisiae TMA29 (YMR226C) gene or a homolog thereof. In one embodiment,the homolog may be selected from the group consisting ofVanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomyces castellii (SEQID NO: 3), Candida glabrata (SEQ ID NO: 4), Saccharomyces bayanus (SEQID NO: 5), Zygosaccharomyces rouxii (SEQ ID NO: 6), Kluyveromyces lactis(SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8), Saccharomyces kluyveri(SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQ ID NO: 10),Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQ ID NO: 12),Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQ ID NO: 14),Candida dubliniensis (SEQ ID NO: 15), Candida albicans (SEQ ID NO: 16),Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis (SEQ ID NO:18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger (SEQ ID NO:20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomyces pombe (SEQID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).

In another embodiment, the invention is directed to a recombinantmicroorganism for producing isobutanol, wherein said recombinantmicroorganism comprises an isobutanol producing metabolic pathway andwherein said microorganism is engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofisobutyraldehyde to isobutyrate. In some embodiments, the enzymecatalyzing the conversion of isobutyraldehyde to isobutyrate is analdehyde dehydrogenase. In an exemplary embodiment, the aldehydedehydrogenase is the S. cerevisiae aldehyde dehydrogenase ALD6 (SEQ IDNO: 25) or a homolog or variant thereof. In one embodiment, the homologis selected from the group consisting of Saccharomyces castelli (SEQ IDNO: 26), Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ IDNO: 28), Kluyveromyces lactis (SEQ ID NO: 29), Kluyveromycesthermotolerans (SEQ ID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31),Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiaeJAY291 (SEQ ID NO: 33), Saccharomyces cerevisiae EC1118 (SEQ ID NO: 34),Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomycescerevisiae AWR11631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a(SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38), Kluyveromycesmarxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40),and Schizosaccharomyces pombe (SEQ ID NO: 41).

In yet another embodiment, the invention is directed to a recombinantmicroorganism for producing isobutanol, wherein said recombinantmicroorganism comprises an isobutanol producing metabolic pathway andwherein said microorganism is (i) engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofacetolactate to DH2MB and (ii) engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofisobutyraldehyde to isobutyrate. In some embodiments, the enzymecatalyzing the conversion of acetolactate to DH2MB is a 3-ketoacidreductase (3-KAR). In a specific embodiment, the 3-ketoacid reductase isencoded by the S. cerevisiae TMA29 (YMR226C) gene or a homolog orvariant thereof. In one embodiment, the homolog is selected from thegroup consisting of Vanderwaltomzyma polyspora (SEQ ID NO: 2),Saccharomyces castelli i (SEQ ID NO: 3), Candida glabrata (SEQ ID NO:4), Saccharomyces bayanus (SEQ ID NO: 5), Zygosaccharomyces rouxii (SEQID NO: 6), Kluyveromyces lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ IDNO: 8), Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromycesthermotolerans (SEQ ID NO: 10), Kluyveromyces waltii (SEQ ID NO: 11),Pichia stipitis (SEQ ID NO: 12), Debaromyces hansenii (SEQ ID NO: 13),Pichia pastoris (SEQ ID NO: 14), Candida dubliniensis (SEQ ID NO: 15),Candida albicans (SEQ ID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17),Issatchenkia orientalis (SEQ ID NO: 18), Aspergillus nidulans (SEQ IDNO: 19), Aspergillus niger (SEQ ID NO: 20), Neurospora crassa (SEQ IDNO: 21), Schizosaccharomyces pombe (SEQ ID NO: 22), and Kluyveromycesmarxianus (SEQ ID NO: 23). In some embodiments, the enzyme catalyzingthe conversion of isobutyraldehyde to isobutyrate is an aldehydedehydrogenase. In a specific embodiment, the aldehyde dehydrogenase isthe S. cerevisiae aldehyde dehydrogenase ALD6 (SEQ ID NO: 25) or ahomolog or variant thereof. In one embodiment, the homolog is selectedfrom the group consisting of Saccharomyces castelli (SEQ ID NO: 26),Candida glabrata (SEQ ID NO: 27), Saccharomyces bayanus (SEQ ID NO: 28),Kluyveromyces lactis (SEQ ID NO: 29), Kluyveromyces thermotolerans (SEQID NO: 30), Kluyveromyces waltii (SEQ ID NO: 31), Saccharomycescerevisiae YJ789 (SEQ ID NO: 32), Saccharomyces cerevisiae JAY291 (SEQID NO: 33), Saccharomyces cerevisiae EC1118 (SEQ ID NO: 34),Saccharomyces cerevisiae DBY939 (SEQ ID NO: 35), Saccharomycescerevisiae AWR11631 (SEQ ID NO: 36), Saccharomyces cerevisiae RM11-1a(SEQ ID NO: 37), Pichia pastoris (SEQ ID NO: 38), Kluyveromycesmarxianus (SEQ ID NO: 39), Schizosaccharomyces pombe (SEQ ID NO: 40),and Schizosaccharomyces pombe (SEQ ID NO: 41).

In one embodiment, the isobutanol producing metabolic pathway comprisesat least one exogenous gene that catalyzes a step in the conversion ofpyruvate to isobutanol. In another embodiment, the isobutanol producingmetabolic pathway comprises at least two exogenous genes that catalyzesteps in the conversion of pyruvate to isobutanol. In yet anotherembodiment, the isobutanol producing metabolic pathway comprises atleast three exogenous genes that catalyze steps in the conversion ofpyruvate to isobutanol. In yet another embodiment, the isobutanolproducing metabolic pathway comprises at least four exogenous genes thatcatalyze steps in the conversion of pyruvate to isobutanol. In yetanother embodiment, the isobutanol producing metabolic pathway comprisesat five exogenous genes that catalyze steps in the conversion ofpyruvate to isobutanol.

In one embodiment, one or more of the isobutanol pathway genes encodesan enzyme that is localized to the cytosol. In one embodiment, therecombinant microorganisms comprise an isobutanol producing metabolicpathway with at least one isobutanol pathway enzyme localized in thecytosol. In another embodiment, the recombinant microorganisms comprisean isobutanol producing metabolic pathway with at least two isobutanolpathway enzymes localized in the cytosol. In yet another embodiment, therecombinant microorganisms comprise an isobutanol producing metabolicpathway with at least three isobutanol pathway enzymes localized in thecytosol. In yet another embodiment, the recombinant microorganismscomprise an isobutanol producing metabolic pathway with at least fourisobutanol pathway enzymes localized in the cytosol. In an exemplaryembodiment, the recombinant microorganisms comprise an isobutanolproducing metabolic pathway with five isobutanol pathway enzymeslocalized in the cytosol. Isobutanol producing metabolic pathways inwhich one or more genes are localized to the cytosol are described incommonly owned and co-pending U.S. application Ser. No. 12/855,276,which is herein incorporated by reference in its entirety for allpurposes.

Expression of Modified Alcohol Dehydrogenases in the Production ofIsobutanol

Another strategy described herein for reducing the production of theby-product isobutyrate is to increase the activity and/or expression ofan alcohol dehydrogenase (ADH) responsible for the conversion ofisobutyraldehyde to isobutanol. This strategy prevents competition byendogenous enzymes for the isobutanol pathway intermediate,isobutyraldehyde. An increase in the activity and/or expression of ADHmay be achieved by various means. For example, ADH activity can beincreased by utilizing a promoter with increased promoter strength or byincreasing the copy number of the alcohol dehydrogenase gene.

In alternative embodiments, the production of the by-product isobutyratemay be reduced by utilizing an ADH with increased specific activity forisobutyraldehyde. Such ADH enzymes with increased specific activity forisobutyraldehyde may be identified in nature, or may result frommodifications to the ADH enzyme, such as the modifications describedherein. In some embodiments, these modifications will produce a decreasein the Michaelis-Menten constant (K_(M)) for isobutyraldehyde. Throughthe use of such modified ADH enzymes, competition by endogenous enzymesfor isobutyraldehyde is further limited. In one embodiment, theisobutyrate yield (mol isobutyrate per mol glucose) in a recombinantmicroorganism comprising a modified ADH as described herein is less thanabout 5%. In another embodiment, the isobutyrate yield (mol isobutyrateper mol glucose) in a recombinant microorganism comprising a modifiedADH as described herein is less than about 1%. In yet anotherembodiment, the isobutyrate yield (mol isobutyrate per mol glucose) in arecombinant microorganism comprising a modified ADH as described hereinis less than about 0.5%, less than about 0.1%, less than about 0.05%, orless than about 0.01%.

Further, by utilizing a modified ADH enzyme, the present inventors mayestablish a situation in which the forward reaction (i.e. theisobutyraldehyde conversion to isobutanol) is the favored reaction overthe reverse reaction (i.e. the conversion of isobutanol toisobutyraldehyde).

The strategies described above generally lead to a decrease inisobutyrate yield, which is accompanied by an increase in isobutanolyield. Hence, the above strategies are useful for decreasing theisobutyrate yield and/or titer and for increasing the ratio ofisobutanol yield over isobutyrate yield.

Accordingly, in one aspect, the present application describes thegeneration of modified ADHs with enhanced activity that can facilitateimproved isobutanol production when co-expressed with the remaining fourisobutanol pathway enzymes. In one embodiment according to this aspect,the present application is directed to recombinant microorganismscomprising one or more modified ADHs. In one embodiment, the recombinantmicroorganism is further engineered to reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion ofacetolactate to DH2MB as described herein. In another embodiment, therecombinant microorganism is further engineered to reduce or eliminatethe expression or activity of an enzyme catalyzing the conversion ofisobutyraldehyde to isobutyrate as described herein.

In addition to the isobutanol biosynthetic pathway, other biosyntheticpathways utilize ADH enzymes for the conversion of an aldehyde to analcohol. For example, ADH enzymes convert various aldehydes to alcoholsas part of biosynthetic pathways for the production of 1-propanol,2-propanol, 1-butanol, 2-butanol, 1-pentanol, 2-methyl-1-butanol, 3- andmethyl-1-butanol.

As used herein, the terms “ADH” or “ADH enzyme” or “alcoholdehydrogenase” are used interchangeably herein to refer to an enzymethat catalyzes the conversion of isobutyraldehyde to isobutanol. ADHsequences are available from a vast array of microorganisms, including,but not limited to, L. lactis (SEQ ID NO: 175), Streptococcuspneumoniae, Staphylococcus aureus, and Bacillus cereus. ADH enzymesmodifiable by the methods of the present invention include, but are notlimited to those, disclosed in commonly owned and co-pending U.S. PatentPublication No. 2010/0143997. A representative list of ADH enzymesmodifiable by the methods described herein can be found in Table 97.

Modified ADH Enzymes

In accordance with the invention, any number of mutations can be made tothe ADH enzymes, and in one embodiment, multiple mutations can be madeto result in an increased ability to convert isobutyraldehyde toisobutanol. Such mutations include point mutations, frame shiftmutations, deletions, and insertions, with one or more (e.g., one, two,three, four, five, or six, etc.) point mutations preferred. In anexemplary embodiment, the modified ADH enzyme comprises one or moremutations at positions corresponding to amino acids selected from: (a)tyrosine 50 of the L. lactis AdhA (SEQ ID NO: 185); (b) glutamine 77 ofthe L. lactis AdhA (SEQ ID NO: 185); (c) valine 108 of the L. lactisAdhA (SEQ ID NO: 185); (d) tyrosine 113 of the L. lactis AdhA (SEQ IDNO: 185); (e) isoleucine 212 of the L. lactis AdhA (SEQ ID NO: 185); and(f) leucine 264 of the L. lactis AdhA (SEQ ID NO: 185), wherein AdhA(SEQ ID NO: 185) is encoded by the L. lactis alcohol dehydrogenase (ADH)gene adhA (SEQ ID NO: 184) or a codon-optimized version thereof (SEQ IDNO: 206).

Mutations may be introduced into the ADH enzymes of the presentinvention using any methodology known to those skilled in the art.Mutations may be introduced randomly by, for example, conducting a PCRreaction in the presence of manganese as a divalent metal ion cofactor.Alternatively, oligonucleotide directed mutagenesis may be used tocreate the modified ADH enzymes which allows for all possible classes ofbase pair changes at any determined site along the encoding DNAmolecule. In general, this technique involves annealing anoligonucleotide complementary (except for one or more mismatches) to asingle stranded nucleotide sequence coding for the ADH enzyme ofinterest. The mismatched oligonucleotide is then extended by DNApolymerase, generating a double-stranded DNA molecule which contains thedesired change in sequence in one strand. The changes in sequence can,for example, result in the deletion, substitution, or insertion of anamino acid. The double-stranded polynucleotide can then be inserted intoan appropriate expression vector, and a mutant or modified polypeptidecan thus be produced. The above-described oligonucleotide directedmutagenesis can, for example, be carried out via PCR.

Enzymes for use in the compositions and methods of the invention includeany enzyme having the ability to convert isobutyraldehyde to isobutanol.Such enzymes include, but are not limited to, the L. lactis AdhA, the S.pneumoniae AdhA, the S. aureus AdhA, and the Bacillus cereus AdhA,amongst others. Additional ADH enzymes modifiable by the methods of thepresent invention include, but are not limited to those, disclosed incommonly owned and co-pending U.S. Patent Publication No. 2010/0143997.A representative list of ADH enzymes modifiable by the methods describedherein can be found in Table 16. As will be understood by one ofordinary skill in the art, modified ADH enzymes may be obtained byrecombinant or genetic engineering techniques that are routine andwell-known in the art. Modified ADH enzymes can, for example, beobtained by mutating the gene or genes encoding the ADH enzyme ofinterest by site-directed or random mutagenesis. Such mutations mayinclude point mutations, deletion mutations, and insertional mutations.For example, one or more point mutations (e.g., substitution of one ormore amino acids with one or more different amino acids) may be used toconstruct modified ADH enzymes of the invention.

The invention further includes homologous ADH enzymes which are 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% identical at the amino acid level to a wild-type ADH enzyme (e.g.,L. lactis AdhA or E. coli AdhA) and exhibit an increased ability toconvert isobutyraldehyde to isobutanol. Also included within theinvention are ADH enzymes, which are 50%, 60%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, or 99% identical at the amino acid level to an ADHenzyme comprising the amino acid sequence set out in SEQ ID NO: 185 andexhibit an increased ability to convert isobutyraldehyde to isobutanolas compared to the unmodified wild-type enzyme. The invention alsoincludes nucleic acid molecules, which encode the above-described ADHenzymes.

The invention also includes fragments of ADH enzymes which comprise atleast 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 aminoacid residues and retain one or more activities associated with ADHenzymes. Such fragments may be obtained by deletion mutation, byrecombinant techniques that are routine and well-known in the art, or byenzymatic digestion of the ADH enzyme(s) of interest using any of anumber of well-known proteolytic enzymes. The invention further includesnucleic acid molecules, which encode the above described modified ADHenzymes and ADH enzyme fragments.

By a protein or protein fragment having an amino acid sequence at least,for example, 50% “identical” to a reference amino acid sequence, it isintended that the amino acid sequence of the protein is identical to thereference sequence except that the protein sequence may include up to 50amino acid alterations per each 100 amino acids of the amino acidsequence of the reference protein. In other words, to obtain a proteinhaving an amino acid sequence at least 50% identical to a referenceamino acid sequence, up to 50% of the amino acid residues in thereference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 50% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino (N—) and/or carboxy (C—) terminal positions of the reference aminoacid sequence and/or anywhere between those terminal positions,interspersed either individually among residues in the referencesequence and/or in one or more contiguous groups within the referencesequence. As a practical matter, whether a given amino acid sequence is,for example, at least 50% identical to the amino acid sequence of areference protein can be determined conventionally using known computerprograms such as those described above for nucleic acid sequenceidentity determinations, or using the CLUSTAL W program (Thompson, J.D., et al., Nucleic Acids Res. 22:4673 4680 (1994)).

In one aspect, amino acid substitutions are made at one or more of theabove identified positions (i.e., amino acid positions equivalent orcorresponding to Y50, Q77, V108, Y113, I212, or L264 of L. lactis AdhA(SEQ ID NO: 185)). Thus, the amino acids at these positions may besubstituted with any other amino acid including Ala, Asn, Arg, Asp, Cys,Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr,and Val. A specific example of a ADH enzyme which exhibits an increasedability to convert isobutyraldehyde to isobutanol is an ADH in which (1)the tyrosine at position 50 has been replaced with a phenylalanine ortryptophan residue, (2) the glutamine at position 77 has been replacedwith an arginine or serine residue, (3) the valine at position 108 hasbeen replaced with a serine or alanine residue, (4) the tyrosine atposition 113 has been replaced with a phenylalanine or glycine residue,(5), the isoleucine at position 212 has been replaced with a threonineor valine residue, and/or (6) the leucine at position 264 is replacedwith a valine residue.

Polypeptides having the ability to convert isobutyraldehyde toisobutanol for use in the invention may be isolated from their naturalprokaryotic or eukaryotic sources according to standard procedures forisolating and purifying natural proteins that are well-known to one ofordinary skill in the art (see, e.g., Houts, G. E., et al., J. Virol.29:517 (1979)). In addition, polypeptides having the ability to convertisobutyraldehyde to isobutanol may be prepared by recombinant DNAtechniques that are familiar to one of ordinary skill in the art (see,e.g., Kotewicz, M. L., et al., Nucl. Acids Res. 16:265 (1988); Soltis,D. A., and Skalka, A. M., Proc. Natl. Acad. Sci. USA 85:3372 3376(1988)).

In one aspect of the invention, modified ADH enzymes are made byrecombinant techniques. To clone a gene or other nucleic acid moleculeencoding an ADH enzyme which will be modified in accordance with theinvention, isolated DNA which contains the ADH enzyme gene or openreading frame may be used to construct a recombinant DNA library. Anyvector, well known in the art, can be used to clone the ADH enzyme ofinterest. However, the vector used must be compatible with the host inwhich the recombinant vector will be transformed.

Prokaryotic vectors for constructing the plasmid library includeplasmids such as those capable of replication in E. coli such as, forexample, pBR322, ColE1, pSC101, pUC-vectors (pUC18, pUC19, etc.: In:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982); and Sambrook et al., In:Molecular Cloning A Laboratory Manual (2d ed.) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989)). Bacillus plasmidsinclude pC194, pUB110, pE194, pC221, pC217, etc. Such plasmids aredisclosed by Glyczan, T. In: The Molecular Biology Bacilli, AcademicPress, York (1982), 307 329. Suitable Streptomyces plasmids includepIJ101 (Kendall et al., J. Bacteriol. 169:4177 4183 (1987)). Pseudomonasplasmids are reviewed by John et al., (Rad. Insec. Dis. 8:693 704(1986)), and Igaki, (Jpn. J. Bacteriol. 33:729 742 (1978)). Broad-hostrange plasmids or cosmids, such as pCP13 (Darzins and Chakrabarty, J.Bacteriol. 159:9 18 (1984)) can also be used for the present invention.

Suitable hosts for cloning the ADH nucleic acid molecules of interestare prokaryotic hosts. One example of a prokaryotic host is E. coli.However, the desired ADH nucleic acid molecules of the present inventionmay be cloned in other prokaryotic hosts including, but not limited to,hosts in the genera Escherichia, Bacillus, Streptomyces, Pseudomonas,Salmonella, Serratia, and Proteus.

Eukaryotic hosts for cloning and expression of the ADH enzyme ofinterest include yeast and fungal cells. A particularly preferredeukaryotic host is yeast. Expression of the desired ADH enzyme in sucheukaryotic cells may require the use of eukaryotic regulatory regionswhich include eukaryotic promoters. Cloning and expressing the ADHnucleic acid molecule in eukaryotic cells may be accomplished by wellknown techniques using well known eukaryotic vector systems.

In accordance with the invention, one or more mutations may be made inany ADH enzyme of interest in order to increase the ability of theenzyme to convert isobutyraldehyde to isobutanol, or confer otherproperties described herein upon the enzyme, in accordance with theinvention. Such mutations include point mutations, frame shiftmutations, deletions, and insertions. Preferably, one or more pointmutations, resulting in one or more amino acid substitutions, are usedto produce ADH enzymes having an enhanced ability to convertisobutyraldehyde to isobutanol. In a preferred aspect of the invention,one or more mutations at positions equivalent or corresponding toposition Y50 (e.g., Y50W or Y50F), Q77 (e.g., Q77S or Q77R), V108 (e.g.V108S or V108A), Y113 (e.g., Y113F or Y113G), 1212 (e.g., I212T orI212V), and/or L264 (e.g. L264V) of the L. lactis AdhA (SEQ ID NO: 185)enzyme may be made to produce the desired result in other ADH enzymes ofinterest.

The corresponding positions of the ADH enzymes identified herein (e.g.the L. lactis AdhA of SEQ ID NO: 185) may be readily identified forother ADH enzymes by one of skill in the art. Thus, given the definedregion and the assays described in the present application, one withskill in the art can make one or a number of modifications, which wouldresult in an increased ability to convert isobutyraldehyde to isobutanolin any ADH enzyme of interest.

In a preferred embodiment, the modified ADH enzymes have from 1 to 6amino acid substitutions selected from positions corresponding to Y50,Q77, V108, Y113, I212, or L264 as compared to the wild-type ADH enzymes.In other embodiments, the modified ADH enzymes have additional aminoacid substitutions at other positions as compared to the respectivewild-type ADH enzymes. Thus, modified ADH enzymes may have at leastabout 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40 different residues in other positions as compared to therespective wild-type ADH enzymes. As will be appreciated by those ofskill in the art, the number of additional positions that may have aminoacid substitutions will depend on the wild-type ADH enzyme used togenerate the variants. Thus, in some instances, up to 50 differentpositions may have amino acid substitutions.

It is understood that various microorganisms can act as “sources” forgenetic material encoding ADH enzymes suitable for use in a recombinantmicroorganism provided herein. For example, In addition, genes encodingthese enzymes can be identified from other fungal and bacterial speciesand can be expressed for the modulation of this pathway. A variety oforganisms could serve as sources for these enzymes, including, but notlimited to, Lactococcus sp., including L. lactis, Lactobacillus sp.,including L. brevis, L. buchneri, L. hilgardii, L. fermentum, L.reuteri, L. vaginalis, L. antri, L. oris, and L. coleohominis,Pediococcus sp., including P. acidilactici, Bacillus sp., including B.cereus, B. thuringiensis, B. coagulans, B. anthracis, B.weihenstephanensis, B. mycoides, and B. amyloliquefaciens, Leptotrichiasp., including L. goodfellowii, L. buccalis, and L. hofstadii,Actinobacillus sp., including A. pleuropneumoniae, Streptococcus sp.,including S. sanguinis, S. parasanguinis, S. gordonii, S. pneumoniae,and S. mitis, Streptobacillus sp., including S. moniliformis,Staphylococcus sp., including S. aureus, Eikenella sp., including E.corrodens, Weissella sp., including W. paramesenteroides, Kingella sp.,including K. oralis, and Rothia sp., including R. dentocariosa, andExiguobacterium sp.

The nucleotide sequences for several ADH enzymes are known. Forinstance, the sequences of ADH enzymes are available from a vast arrayof microorganisms, including, but not limited to, L. lactis (SEQ ID NO:185), S. pneumoniae, S. aureus, and Bacillus cereus. ADH enzymesmodifiable by the methods of the present invention include, but are notlimited to those, disclosed in commonly owned and co-pending U.S. PatentPublication No. 2010/0143997. A representative list of ADH enzymesmodifiable by the methods described herein can be found in Table 97.

In addition, any method can be used to identify genes that encode forADH enzymes with a specific activity. Generally, homologous or analogousgenes with similar activity can be identified by functional, structural,and/or genetic analysis. In most cases, homologous or analogous geneswith similar activity will have functional, structural, or geneticsimilarities. Techniques known to those skilled in the art may besuitable to identify homologous genes and homologous enzymes. Generally,analogous genes and/or analogous enzymes can be identified by functionalanalysis and will have functional similarities. Techniques known tothose skilled in the art may be suitable to identify analogous genes andanalogous enzymes. For example, to identify homologous or analogousgenes, proteins, or enzymes, techniques may include, but not limited to,cloning a gene by PCR using primers based on a published sequence of agene/enzyme or by degenerate PCR using degenerate primers designed toamplify a conserved region among a gene. Further, one skilled in the artcan use techniques to identify homologous or analogous genes, proteins,or enzymes with functional homology or similarity. Techniques includeexamining a cell or cell culture for the catalytic efficiency or thespecific activity of an enzyme through in vitro enzyme assays for saidactivity, then isolating the enzyme with said activity throughpurification, determining the protein sequence of the enzyme throughtechniques such as Edman degradation, design of PCR primers to thelikely nucleic acid sequence, amplification of said DNA sequence throughPCR, and cloning of said nucleic acid sequence. To identify homologousor analogous genes with similar activity, techniques also includecomparison of data concerning a candidate gene or enzyme with databasessuch as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme may beidentified within the above mentioned databases in accordance with theteachings herein. Furthermore, enzymatic activity can be determinedphenotypically.

Methods of Making ADH Enzymes with Enhanced Catalytic Efficiency

The present invention further provides methods of engineering ADHenzymes to enhance their catalytic efficiency.

One approach to increasing the catalytic efficiency of ADH enzymes is bysaturation mutagenesis with NNK libraries. These libraries may bescreened for increases in catalytic efficiency in order to identify,which single mutations contribute to an increased ability to convertisobutyraldehyde to isobutanol. Combinations of mutations ataforementioned residues may be investigated by any method. For example,a combinatorial library of mutants may be designed based on the resultsof the saturation mutagenesis studies.

Another approach is to use random oligonucleotide mutagenesis togenerate diversity by incorporating random mutations, encoded on asynthetic oligonucleotide, into the enzyme. The number of mutations inindividual enzymes within the population may be controlled by varyingthe length of the target sequence and the degree of randomization duringsynthesis of the oligonucleotides. The advantages of this more definedapproach are that all possible amino acid mutations and also coupledmutations can be found.

If the best variants from the experiments described above do not displaysufficient activity, directed evolution via error-prone PCR may be usedto obtain further improvements. Error-prone PCR mutagenesis of the ADHenzyme may be performed followed by screening for ADH activity.

Enhanced ADH Catalytic Efficiency

In one aspect, the catalytic efficiency of the modified ADH enzyme isenhanced. As used herein, the phrase “catalytic efficiency” refers tothe property of the ADH enzyme that allows it to convertisobutyraldehyde to isobutanol.

In one embodiment, the catalytic efficiency of the modified ADH isenhanced as compared to the wild-type or parental ADH. Preferably, thecatalytic efficiency of the modified ADH enzyme is enhanced by at leastabout 5% as compared to the wild-type or parental ADH. More preferably,the catalytic efficiency of the modified ADH enzyme is enhanced by atleast about 15% as compared to the wild-type or parental ADH. Morepreferably, the catalytic efficiency of the modified ADH enzyme isenhanced by at least about 25% as compared to the wild-type or parentalADH. More preferably, the catalytic efficiency of the modified ADHenzyme is enhanced by at least about 50% as compared to the wild-type orparental ADH. More preferably, the catalytic efficiency of the modifiedADH enzyme is enhanced by at least about 75% as compared to thewild-type or parental ADH. More preferably, the catalytic efficiency ofthe modified ADH enzyme is enhanced by at least about 100% as comparedto the wild-type or parental ADH. More preferably, the catalyticefficiency of the modified ADH enzyme is enhanced by at least about 200%as compared to the wild-type or parental ADH. More preferably, thecatalytic efficiency of the modified ADH enzyme is enhanced by at leastabout 500% as compared to the wild-type or parental ADH. Morepreferably, the catalytic efficiency of the modified ADH enzyme isenhanced by at least about 1000% as compared to the wild-type orparental ADH. More preferably, the catalytic efficiency of the modifiedADH enzyme is enhanced by at least about 2000% as compared to thewild-type or parental ADH. More preferably, the catalytic efficiency ofthe modified ADH enzyme is enhanced by at least about 3000% as comparedto the wild-type or parental ADH. Most preferably, the catalyticefficiency of the modified ADH enzyme is enhanced by at least about3500% as compared to the wild-type or parental ADH.

Gene Expression of Modified ADH Enzymes

Provided herein are methods for the expression of one or more of themodified ADH enzyme genes involved the production of beneficialmetabolites and recombinant DNA expression vectors useful in the method.Thus, included within the scope of the disclosure are recombinantexpression vectors that include such nucleic acids. The term expressionvector refers to a nucleic acid that can be introduced into a hostmicroorganism or cell-free transcription and translation system. Anexpression vector can be maintained permanently or transiently in amicroorganism, whether as part of the chromosomal or other DNA in themicroorganism or in any cellular compartment, such as a replicatingvector in the cytoplasm. An expression vector also comprises a promoterthat drives expression of an RNA, which typically is translated into apolypeptide in the microorganism or cell extract. For efficienttranslation of RNA into protein, the expression vector also typicallycontains a ribosome-binding site sequence positioned upstream of thestart codon of the coding sequence of the gene to be expressed. Otherelements, such as enhancers, secretion signal sequences, transcriptiontermination sequences, and one or more marker genes by which hostmicroorganisms containing the vector can be identified and/or selected,may also be present in an expression vector. Selectable markers, i.e.,genes that confer antibiotic resistance or sensitivity, are used andconfer a selectable phenotype on transformed cells when the cells aregrown in an appropriate selective medium.

The various components of an expression vector can vary widely,depending on the intended use of the vector and the host cell(s) inwhich the vector is intended to replicate or drive expression.Expression vector components suitable for the expression of genes andmaintenance of vectors in E. coli, yeast, Streptomyces, and othercommonly used cells are widely known and commercially available. Forexample, suitable promoters for inclusion in the expression vectors ofthe disclosure include those that function in eukaryotic or prokaryotichost microorganisms. Promoters can comprise regulatory sequences thatallow for regulation of expression relative to the growth of the hostmicroorganism or that cause the expression of a gene to be turned on oroff in response to a chemical or physical stimulus. For E. coli andcertain other bacterial host cells, promoters derived from genes forbiosynthetic enzymes, antibiotic-resistance conferring enzymes, andphage proteins can be used and include, for example, the galactose,lactose (lac), maltose, tryptophan (trp), beta-lactamase (bla),bacteriophage lambda PL, and T5 promoters. In addition, syntheticpromoters, such as the tac promoter (U.S. Pat. No. 4,551,433), can alsobe used. For E. coli expression vectors, it is useful to include an E.coli origin of replication, such as from pUC, p1P, p1, and pBR.

Thus, recombinant expression vectors contain at least one expressionsystem, which, in turn, is composed of at least a portion of abiosynthetic gene coding sequences operably linked to a promoter andoptionally termination sequences that operate to effect expression ofthe coding sequence in compatible host cells. The host cells aremodified by transformation with the recombinant DNA expression vectorsof the disclosure to contain the expression system sequences either asextrachromosomal elements or integrated into the chromosome.

Moreover, methods for expressing a polypeptide from a nucleic acidmolecule that are specific to a particular microorganism (i.e. a yeastmicroorganism) are well known. For example, nucleic acid constructs thatare used for the expression of heterologous polypeptides withinKluyveromyces and Saccharomyces are well known (see, e.g., U.S. Pat.Nos. 4,859,596 and 4,943,529, each of which is incorporated by referenceherein in its entirety for Kluyveromyces and, e.g., Gellissen et al.,Gene 190(1):87-97 (1997) for Saccharomyces. Yeast plasmids have aselectable marker and an origin of replication, also known asAutonomously Replicating Sequences (ARS). In addition certain plasmidsmay also contain a centromeric sequence. These centromeric plasmids aregenerally a single or low copy plasmid. Plasmids without a centromericsequence and utilizing either a 2 micron (S. cerevisiae) or 1.6 micron(K. lactis) replication origin are high copy plasmids. The selectablemarker can be either prototrophic, such as HIS3, TRP1, LEU2, URA3 orADE2, or antibiotic resistance, such as, bar, ble, hph, or kan.

A nucleic acid of the disclosure can be amplified using cDNA, mRNAsynthetic DNA, or alternatively, genomic DNA, as a template andappropriate oligonucleotide primers according to standard PCRamplification techniques and those procedures described in the Examplessection below. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to nucleotide sequences canbe prepared by standard synthetic techniques, e.g., using an automatedDNA synthesizer.

It is also understood that an isolated nucleic acid molecule encoding apolypeptide homologous to the enzymes described herein can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence encoding the particular polypeptide, suchthat one or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intothe polynucleotide by standard techniques, such as site-directedmutagenesis and PCR-mediated mutagenesis. In contrast to those positionswhere it may be desirable to make a non-conservative amino acidsubstitutions (see above), in some positions it is preferable to makeconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

Although the effect of an amino acid change varies depending uponfactors such as phosphorylation, glycosylation, intra-chain linkages,tertiary structure, and the role of the amino acid in the active site ora possible allosteric site, it is generally preferred that thesubstituted amino acid is from the same group as the amino acid beingreplaced. To some extent the following groups contain amino acids, whichare interchangeable: the basic amino acids lysine, arginine, andhistidine; the acidic amino acids aspartic and glutamic acids; theneutral polar amino acids serine, threonine, cysteine, glutamine,asparagine and, to a lesser extent, methionine; the nonpolar aliphaticamino acids glycine, alanine, valine, isoleucine, and leucine (however,because of size, glycine and alanine are more closely related andvaline, isoleucine and leucine are more closely related); and thearomatic amino acids phenylalanine, tryptophan, and tyrosine. Inaddition, although classified in different categories, alanine, glycine,and serine seem to be interchangeable to some extent, and cysteineadditionally fits into this group, or may be classified with the polarneutral amino acids.

Methods in General Identification of 3-Ketoacid Reductase Homologs

Any method can be used to identify genes that encode for enzymes with3-ketoacid reductase activity, including, but not limited to S.cerevisiae TMA29. Generally, genes that are homologous or similar to3-ketoacid reductases such as TMA29 can be identified by functional,structural, and/or genetic analysis. In most cases, homologous orsimilar genes and/or homologous or similar enzymes will have functional,structural, or genetic similarities.

The S. cerevisiae gene TMA29 is also known as YMR226C. The open readingframe (ORF) YMR226C is found on the S. cerevisiae Chromosome XIII atpositions 722395 . . . 721592. The chromosomal location of YMR226C is aregion that is highly syntenic to chromosomes in many related yeast[Byrne, K. P. and K. H. Wolfe (2005) “The Yeast Gene Order Browser:combining curated homology and syntenic context reveals gene fate inpolyploid species.” Genome Res. 15(10):1456-61. Scannell, D. R., K. P.Byrne, J. L. Gordon, S. Wong, and K. H. Wolfe (2006) “Multiple rounds ofspeciation associated with reciprocal gene loss in polyploidy yeasts.”Nature 440: 341-5. Scannell, D. R., A. C. Frank, G. C. Conant, K. P.Byrne, M. Woolfit, and K. H. Wolfe (2007)” Independent sorting-out ofthousands of duplicated gene pairs in two yeast species descended from awhole-genome duplication.” Proc Natl Acad Sci USA 104: 8397-402.]

For example, locations of the syntenic versions of YMR226C from otheryeast species can be found on Chromosome 13 in Candida glabrata,Chromosome 1 in Zygosaccharomyces rouxii, Chromosome 2 in K. lactis,Chromosome 6 in Ashbya gossypii, Chromosome 8 in S. kluyveri, Chromosome4 in K. thermotolerance and Chromosome 8 from the inferred ancestralyeast species [Gordon, J. L., K. P. Byrne, and K. H. Wolfe (2009)“Additions, losses, and rearrangements on the evolutionary route from areconstructed ancestor to the modern Saccharomyces cerevisiae genome.”PLoS Genet. 5: e1000485.]

Using this syntenic relationship, species-specific versions of this geneare readily identified and examples can be found in Table 4.

TABLE 4 YMR226C and homologs thereof. Species Gene Name SEQ ID NO: S.cerevisiae YMR226C 1 K. polyspora Kpol_1043p53 2 S. castelliiScas_594.12d 3 C. glabrata CAGL0M11242g 4 S. bayanus Sbay_651.2 5 Z.rouxii ZYRO0A05742p 6 K. lactis KLLA0B08371g 7 A. gossypii AFR561Wp 8 S.kluyveri SAKL0H04730g 9 K. thermotolerans KLTH0D13002p 10 K. waltiiKwal_26.9160 11

In addition to synteny, fungal homologs to the S. cerevisiae TMA29 genemay be identified by one skilled in the art through tools such as BLASTand sequence alignment. These other homologs may be deleted in a similarmanner from the respective yeast species to eliminate the accumulationof the 3-hydroxyacid by-product. Examples of homologous proteins can befound in Vanderwaltomzyma polyspora (SEQ ID NO: 2), Saccharomycescastelli i (SEQ ID NO: 3), Candida glabrata (SEQ ID NO: 4),Saccharomyces bayanus (SEQ ID NO: 5), Zygosaccharomyces rouxii (SEQ IDNO: 6), K. lactis (SEQ ID NO: 7), Ashbya gossypii (SEQ ID NO: 8),Saccharomyces kluyveri (SEQ ID NO: 9), Kluyveromyces thermotolerans (SEQID NO: 10), Kluyveromyces waltii (SEQ ID NO: 11), Pichia stipitis (SEQID NO: 12), Debaromyces hansenii (SEQ ID NO: 13), Pichia pastoris (SEQID NO: 14), Candida dubliniensis (SEQ ID NO: 15), Candida albicans (SEQID NO: 16), Yarrowia lipolytica (SEQ ID NO: 17), Issatchenkia orientalis(SEQ ID NO: 18), Aspergillus nidulans (SEQ ID NO: 19), Aspergillus niger(SEQ ID NO: 20), Neurospora crassa (SEQ ID NO: 21), Schizosaccharomycespombe (SEQ ID NO: 22), and Kluyveromyces marxianus (SEQ ID NO: 23).

Techniques known to those skilled in the art may be suitable to identifyadditional homologous genes and homologous enzymes. Generally, analogousgenes and/or analogous enzymes can be identified by functional analysisand will have functional similarities. Techniques known to those skilledin the art may be suitable to identify analogous genes and analogousenzymes. For example, to identify homologous or analogous genes,proteins, or enzymes, techniques may include, but not limited to,cloning a dehydratase gene by PCR using primers based on a publishedsequence of a gene/enzyme or by degenerate PCR using degenerate primersdesigned to amplify a conserved region among dehydratase genes. Further,one skilled in the art can use techniques to identify homologous oranalogous genes, proteins, or enzymes with functional homology orsimilarity. Techniques include examining a cell or cell culture for thecatalytic activity of an enzyme through in vitro enzyme assays for saidactivity (e.g. as described herein or in Kiritani, K. Branched-ChainAmino Acids Methods Enzymology, 1970), then isolating the enzyme withsaid activity through purification, determining the protein sequence ofthe enzyme through techniques such as Edman degradation, design of PCRprimers to the likely nucleic acid sequence, amplification of said DNAsequence through PCR, and cloning of said nucleic acid sequence. Toidentify homologous or similar genes and/or homologous or similarenzymes, analogous genes and/or analogous enzymes or proteins,techniques also include comparison of data concerning a candidate geneor enzyme with databases such as BRENDA, KEGG, or MetaCYC. The candidategene or enzyme may be identified within the above mentioned databases inaccordance with the teachings herein.

Identification of Aldehyde Dehydrogenase Homologs

Any method can be used to identify genes that encode for enzymes withaldehyde dehydrogenase activity, including, but not limited, to the S.cerevisiae ALD6. Generally, genes that are homologous or similar toaldehyde dehydrogenases such as ALD6 can be identified by functional,structural, and/or genetic analysis. In most cases, homologous orsimilar genes and/or homologous or similar enzymes will have functional,structural, or genetic similarities.

The S. cerevisiae gene ALD6 is also known by its systematic nameYPL061W. The open reading frame (ORF) YPL061W is found on the S.cerevisiae Chromosome XVI at positions 432585 . . . 434087. Thechromosomal location of YPL061W is a region that is highly syntenic tochromosomes in many related yeast [Byrne, K. P. and K. H. Wolfe (2005)“The Yeast Gene Order Browser: combining curated homology and synteniccontext reveals gene fate in polyploid species.” Genome Res. 15:1456-61. Scannell, D. R., K. P. Byrne, J. L. Gordon, S. Wong, and K. H.Wolfe (2006) “Multiple rounds of speciation associated with reciprocalgene loss in polyploidy yeasts.” Nature 440: 341-5. Scannell, D. R., A.C. Frank, G. C. Conant, K. P. Byrne, M. Woolfit, and K. H. Wolfe (2007)”Independent sorting-out of thousands of duplicated gene pairs in twoyeast species descended from a whole-genome duplication.” Proc Natl AcadSci USA 104: 8397-402.]

For example, locations of the syntenic versions of YPL061W from otheryeast species can be found on Chromosome 8 in Candida glabrata,Chromosome 5 in K. lactis, Chromosome 5 in K. thermotolerans andChromosome 8 from the inferred ancestral yeast species [Gordon, J. L.,K. P. Byrne, and K. H. Wolfe (2009) “Additions, losses, andrearrangements on the evolutionary route from a reconstructed ancestorto the modern Saccharomyces cerevisiae genome.” PLoS Genet. 5:e1000485.].

Using this syntenic relationship, species-specific versions of this geneare readily identified and examples can be found in Table 5.

TABLE 5 ALD6 and homologs thereof. Species Gene Name SEQ ID NO: S.cerevisiae YPL061W 25 S. castellii Scas_664.24 26 C. glabrataCAGL0H05137g 27 S. bayanus Sbay_623.4 28 K. lactis KLLA0E23057 29 K.thermotolerans KLTH0E12210g 30 K. waltii Kwal_27.119760 31

In addition to synteny, fungal homologs to the S. cerevisiae ALD6 genemay be identified by one skilled in the art through tools such as BLASTand sequence alignment. These other homologs may be deleted in a similarmanner from the respective yeast species to eliminate the accumulationof the aldehyde by-product. Examples of homologous proteins can be foundin Saccharomyces castelli (SEQ ID NO: 26), Candida glabrata (SEQ ID NO:27), Saccharomyces bayanus (SEQ ID NO: 28), Kluyveromyces lactis (SEQ IDNO: 29), Kluyveromyces thermotolerans (SEQ ID NO: 30), Kluyveromyceswaltii (SEQ ID NO: 31), Saccharomyces cerevisiae YJ789 (SEQ ID NO: 32),Saccharomyces cerevisiae JAY291 (SEQ ID NO: 33), Saccharomycescerevisiae EC1118 (SEQ ID NO: 34), Saccharomyces cerevisiae DBY939 (SEQID NO: 35), Saccharomyces cerevisiae AWR11631 (SEQ ID NO: 36),Saccharomyces cerevisiae RM11-1a (SEQ ID NO: 37), Pichia pastoris (SEQID NO: 38), Kluyveromyces marxianus (SEQ ID NO: 39), Schizosaccharomycespombe (SEQ ID NO: 40), and Schizosaccharomyces pombe (SEQ ID NO: 41).

Identification of an ADH or KDH in a Microorganism

Any method can be used to identify genes that encode for enzymes withalcohol dehydrogenase (ADH) or ketoacid dehydrogenase (KDH) activity.Alcohol dehydrogenase (ADH) can catalyze the reversible conversion ofisobutanol to isobutyraldehyde. Ketoacid dehydrogenases (KDH) cancatalyze the conversion of 2-ketoisovalerate to isobutyryl-CoA, whichcan be converted further to isobutyrate by the action of transacetylaseand carboxylic acid kinase enzymes. Generally, genes that are homologousor similar to known alcohol dehydrogenases and ketoacid dehydrogenasescan be identified by functional, structural, and/or genetic analysis. Inmost cases, homologous or similar alcohol dehydrogenase genes and/orhomologous or similar alcohol dehydrogenase enzymes will havefunctional, structural, or genetic similarities. Likewise, homologous orsimilar ketoacid dehydrogenase genes and/or homologous or similarketoacid dehydrogenase enzymes will have functional, structural, orgenetic similarities.

Identification of PDC and GPD in a Yeast Microorganism

Any method can be used to identify genes that encode for enzymes withpyruvate decarboxylase (PDC) activity or glycerol-3-phosphatedehydrogenase (GPD) activity. Suitable methods for the identification ofPDC and GPD are described in commonly owned and co-pending publications,US 2009/0226991 and US 2011/0020889, both of which are hereinincorporated by reference in their entireties for all purposes.

Genetic Insertions and Deletions

Any method can be used to introduce a nucleic acid molecule into yeastand many such methods are well known. For example, transformation andelectroporation are common methods for introducing nucleic acid intoyeast cells. See, e.g., Gietz et al., 1992, Nuc Acids Res. 27: 69-74;Ito et al., 1983, J. Bacteriol. 153: 163-8; and Becker et al., 1991,Methods in Enzymology 194: 182-7.

In an embodiment, the integration of a gene of interest into a DNAfragment or target gene of a yeast microorganism occurs according to theprinciple of homologous recombination. According to this embodiment, anintegration cassette containing a module comprising at least one yeastmarker gene and/or the gene to be integrated (internal module) isflanked on either side by DNA fragments homologous to those of the endsof the targeted integration site (recombinogenic sequences). Aftertransforming the yeast with the cassette by appropriate methods, ahomologous recombination between the recombinogenic sequences may resultin the internal module replacing the chromosomal region in between thetwo sites of the genome corresponding to the recombinogenic sequences ofthe integration cassette. (Orr-Weaver et al., 1981, PNAS USA 78:6354-58).

In an embodiment, the integration cassette for integration of a gene ofinterest into a yeast microorganism includes the heterologous gene underthe control of an appropriate promoter and terminator together with theselectable marker flanked by recombinogenic sequences for integration ofa heterologous gene into the yeast chromosome. In an embodiment, theheterologous gene includes an appropriate native gene desired toincrease the copy number of a native gene(s). The selectable marker genecan be any marker gene used in yeast, including but not limited to,HIS3, TRP1, LEU2, URA3, bar, ble, hph, and kan. The recombinogenicsequences can be chosen at will, depending on the desired integrationsite suitable for the desired application.

In another embodiment, integration of a gene into the chromosome of theyeast microorganism may occur via random integration (Kooistra et al.,2004, Yeast 21: 781-792).

Additionally, in an embodiment, certain introduced marker genes areremoved from the genome using techniques well known to those skilled inthe art. For example, URA3 marker loss can be obtained by plating URA3containing cells in FOA (5-fluoro-orotic acid) containing medium andselecting for FOA resistant colonies (Boeke et al., 1984, Mol. Gen.Genet. 197: 345-47).

The exogenous nucleic acid molecule contained within a yeast cell of thedisclosure can be maintained within that cell in any form. For example,exogenous nucleic acid molecules can be integrated into the genome ofthe cell or maintained in an episomal state that can stably be passed on(“inherited”) to daughter cells. Such extra-chromosomal genetic elements(such as plasmids, mitochondrial genome, etc.) can additionally containselection markers that ensure the presence of such genetic elements indaughter cells. Moreover, the yeast cells can be stably or transientlytransformed. In addition, the yeast cells described herein can contain asingle copy, or multiple copies of a particular exogenous nucleic acidmolecule as described above.

Reduction of Enzymatic Activity

Yeast microorganisms within the scope of the invention may have reducedenzymatic activity such as reduced 3-ketoacid reductase, PDC, ALDH, orglycerol-3-phosphate dehydrogenase (GPD) activity. The term “reduced” asused herein with respect to a particular enzymatic activity refers to alower level of enzymatic activity than that measured in a comparableyeast cell of the same species. The term reduced also refers to theelimination of enzymatic activity as compared to a comparable yeast cellof the same species. Thus, yeast cells lacking 3-ketoacid reductase,PDC, ALDH or glycerol-3-phosphate dehydrogenase (GPD) activity areconsidered to have reduced 3-ketoacid reductase, PDC, ALDH orglycerol-3-phosphate dehydrogenase (GPD) activity since most, if notall, comparable yeast strains have at least some 3-ketoacid reductase,PDC, ALDH, or glycerol-3-phosphate dehydrogenase (GPD) activity. Suchreduced enzymatic activities can be the result of lower enzymeconcentration, lower specific activity of an enzyme, or a combinationthereof. Many different methods can be used to make yeast having reducedenzymatic activity. For example, a yeast cell can be engineered to havea disrupted enzyme-encoding locus using common mutagenesis or knock-outtechnology. See, e.g., Methods in Yeast Genetics (1997 edition), Adams,Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998). Inaddition, certain point-mutation(s) can be introduced which results inan enzyme with reduced activity. Also included within the scope of thisinvention are yeast strains which when found in nature, aresubstantially free of one or more activities selected from 3-ketoacidreductase, PDC, ALDH, or glycerol-3-phosphate dehydrogenase (GPD)activity.

Alternatively, antisense technology can be used to reduce enzymaticactivity. For example, yeast can be engineered to contain a cDNA thatencodes an antisense molecule that prevents an enzyme from being made.The term “antisense molecule” as used herein encompasses any nucleicacid molecule that contains sequences that correspond to the codingstrand of an endogenous polypeptide. An antisense molecule also can haveflanking sequences (e.g., regulatory sequences). Thus antisensemolecules can be ribozymes or antisense oligonucleotides. A ribozyme canhave any general structure including, without limitation, hairpin,hammerhead, or axhead structures, provided the molecule cleaves RNA.

Yeast having a reduced enzymatic activity can be identified using manymethods. For example, yeast having reduced 3-ketoacid reductase, PDC,ALDH, or glycerol-3-phosphate dehydrogenase (GPD) activity can be easilyidentified using common methods, which may include, for example,measuring glycerol formation via liquid chromatography.

Overexpression of Heterologous Genes

Methods for overexpressing a polypeptide from a native or heterologousnucleic acid molecule are well known. Such methods include, withoutlimitation, constructing a nucleic acid sequence such that a regulatoryelement promotes the expression of a nucleic acid sequence that encodesthe desired polypeptide. Typically, regulatory elements are DNAsequences that regulate the expression of other DNA sequences at thelevel of transcription. Thus, regulatory elements include, withoutlimitation, promoters, enhancers, and the like. For example, theexogenous genes can be under the control of an inducible promoter or aconstitutive promoter. Moreover, methods for expressing a polypeptidefrom an exogenous nucleic acid molecule in yeast are well known. Forexample, nucleic acid constructs that are used for the expression ofexogenous polypeptides within Kluyveromyces and Saccharomyces are wellknown (see, e.g., U.S. Pat. Nos. 4,859,596 and 4,943,529, forKluyveromyces and, e.g., Gellissen et al., Gene 190(1):87-97 (1997) forSaccharomyces). Yeast plasmids have a selectable marker and an origin ofreplication. In addition certain plasmids may also contain a centromericsequence. These centromeric plasmids are generally a single or low copyplasmid. Plasmids without a centromeric sequence and utilizing either a2 micron (S. cerevisiae) or 1.6 micron (K. lactis) replication originare high copy plasmids. The selectable marker can be eitherprototrophic, such as HIS3, TRP1, LEU2, URA3 or ADE2, or antibioticresistance, such as, bar, ble, hph, or kan.

In another embodiment, heterologous control elements can be used toactivate or repress expression of endogenous genes. Additionally, whenexpression is to be repressed or eliminated, the gene for the relevantenzyme, protein or RNA can be eliminated by known deletion techniques.

As described herein, any yeast within the scope of the disclosure can beidentified by selection techniques specific to the particular enzymebeing expressed, over-expressed or repressed. Methods of identifying thestrains with the desired phenotype are well known to those skilled inthe art. Such methods include, without limitation, PCR, RT-PCR, andnucleic acid hybridization techniques such as Northern and Southernanalysis, altered growth capabilities on a particular substrate or inthe presence of a particular substrate, a chemical compound, a selectionagent and the like. In some cases, immunohistochemistry and biochemicaltechniques can be used to determine if a cell contains a particularnucleic acid by detecting the expression of the encoded polypeptide. Forexample, an antibody having specificity for an encoded enzyme can beused to determine whether or not a particular yeast cell contains thatencoded enzyme. Further, biochemical techniques can be used to determineif a cell contains a particular nucleic acid molecule encoding anenzymatic polypeptide by detecting a product produced as a result of theexpression of the enzymatic polypeptide. For example, transforming acell with a vector encoding acetolactate synthase and detectingincreased acetolactate concentrations compared to a cell without thevector indicates that the vector is both present and that the geneproduct is active. Methods for detecting specific enzymatic activitiesor the presence of particular products are well known to those skilledin the art. For example, the presence of acetolactate can be determinedas described by Hugenholtz and Starrenburg, 1992, Appl. Micro. Biot.38:17-22.

Increase of Enzymatic Activity

Yeast microorganisms of the invention may be further engineered to haveincreased activity of enzymes (e.g., increased activity of enzymesinvolved in an isobutanol producing metabolic pathway). The term“increased” as used herein with respect to a particular enzymaticactivity refers to a higher level of enzymatic activity than thatmeasured in a comparable yeast cell of the same species. For example,overexpression of a specific enzyme can lead to an increased level ofactivity in the cells for that enzyme. Increased activities for enzymesinvolved in glycolysis or the isobutanol pathway would result inincreased productivity and yield of isobutanol.

Methods to increase enzymatic activity are known to those skilled in theart. Such techniques may include increasing the expression of the enzymeby increased copy number and/or use of a strong promoter, introductionof mutations to relieve negative regulation of the enzyme, introductionof specific mutations to increase specific activity and/or decrease theKm for the substrate, or by directed evolution. See, e.g., Methods inMolecular Biology (vol. 231), ed. Arnold and Georgiou, Humana Press(2003).

Methods of Using Recombinant Microorganisms for High-Yield Fermentations

For a biocatalyst to produce a beneficial metabolite most economically,it is desirable to produce said metabolite at a high yield. Preferably,the only product produced is the desired metabolite, as extra products(i.e. by-products) lead to a reduction in the yield of the desiredmetabolite and an increase in capital and operating costs, particularlyif the extra products have little or no value. These extra products alsorequire additional capital and operating costs to separate theseproducts from the desired metabolite.

In one aspect, the present invention provides a method of producing abeneficial metabolite derived from a recombinant microorganismcomprising a biosynthetic pathway.

In one embodiment, the method includes cultivating a recombinantmicroorganism comprising a biosynthetic pathway which uses a 3-ketoacidas an intermediate in a culture medium containing a feedstock providingthe carbon source until a recoverable quantity of the beneficialmetabolite is produced and optionally, recovering the metabolite. In oneembodiment, the 3-ketoacid intermediate is acetolactate. In an exemplaryembodiment, said recombinant microorganism is engineered to reduce oreliminate the expression or activity of an enzyme catalyzing theconversion of acetolactate to DH2MB. The beneficial metabolite may bederived from any biosynthetic pathway which uses acetolactate asintermediate, including, but not limited to, biosynthetic pathways forthe production of isobutanol, 2-butanol, 1-butanol, 2-butanone,2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid,isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. Ina specific embodiment, the beneficial metabolite is isobutanol. Inanother embodiment, the 3-ketoacid intermediate is2-aceto-2-hydroxybutyrate. In an exemplary embodiment, said recombinantmicroorganism is engineered to reduce or eliminate the expression oractivity of an enzyme catalyzing the conversion of2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrate. Thebeneficial metabolite may be derived from any biosynthetic pathway whichuses 2-aceto-2-hydroxybutyrate as intermediate, including, but notlimited to, biosynthetic pathways for the production of2-methyl-1-butanol, isoleucine, 3-methyl-1-pentanol, 4-methyl-1-hexanol,and 5-methyl-1-heptanol.

In another embodiment, the method includes cultivating a recombinantmicroorganism comprising a biosynthetic pathway which uses an aldehydeas an intermediate in a culture medium containing a feedstock providingthe carbon source until a recoverable quantity of the beneficialmetabolite is produced and optionally, recovering the metabolite. In anexemplary embodiment, said recombinant microorganism is engineered toreduce or eliminate the expression or activity of an enzyme catalyzingthe conversion of an aldehyde to acid by-product. The beneficialmetabolite may be derived from any biosynthetic pathway which uses analdehyde as intermediate, including, but not limited to, biosyntheticpathways for the production of isobutanol, 1-butanol,2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol, 1-pentanol,1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol,and 5-methyl-1-heptanol. In a specific embodiment, the beneficialmetabolite is isobutanol.

In another embodiment, the method includes cultivating a recombinantmicroorganism comprising a biosynthetic pathway which uses acetolactateand an aldehyde as intermediates in a culture medium containing afeedstock providing the carbon source until a recoverable quantity ofthe beneficial metabolite is produced and optionally, recovering themetabolite. In an exemplary embodiment, said recombinant microorganismis engineered to (i) reduce or eliminate the expression or activity ofan enzyme catalyzing the conversion of acetolactate to DH2MB and (ii)reduce or eliminate the expression or activity of an enzyme catalyzingthe conversion of an aldehyde to acid by-product. The beneficialmetabolite may be derived from any biosynthetic pathway which usesacetolactate and an aldehyde as intermediate, including, but not limitedto, biosynthetic pathways for the production of isobutanol, 1-butanol,and 3-methyl-1-butanol. In a specific embodiment, the beneficialmetabolite is isobutanol.

In another embodiment, the method includes cultivating a recombinantmicroorganism comprising a biosynthetic pathway which uses2-aceto-2-hydroxybutyrate and an aldehyde as intermediates in a culturemedium containing a feedstock providing the carbon source until arecoverable quantity of the beneficial metabolite is produced andoptionally, recovering the metabolite. In an exemplary embodiment, saidrecombinant microorganism is engineered to (i) reduce or eliminate theexpression or activity of an enzyme catalyzing the conversion of2-aceto-2-hydroxybutyrate to 2-ethyl-2,3-dihydroxybutyrate and (ii)reduce or eliminate the expression or activity of an enzyme catalyzingthe conversion of an aldehyde to acid by-product. The beneficialmetabolite may be derived from any biosynthetic pathway which uses2-aceto-2-hydroxybutyrate and an aldehyde as intermediate, including,but not limited to, biosynthetic pathways for the production of2-methyl-1-butanol, 3-methyl-1-pentanol, 4-methyl-1-hexanol, and5-methyl-1-heptanol.

In another embodiment, the present invention provides a method ofproducing a beneficial metabolite derived from an alcohol dehydrogenase(ADH)-requiring biosynthetic pathway. In one embodiment, the methodincludes cultivating a recombinant microorganism comprising a modifiedADH described herein in a culture medium containing a feedstockproviding the carbon source until a recoverable quantity of thebeneficial metabolite is produced and optionally, recovering themetabolite. The beneficial metabolite may be derived from anyADH-requiring biosynthetic pathway, including, but not limited to,biosynthetic pathways for the production of 1-propanol, 2-propanol,1-butanol, 2-butanol, 1-pentanol, 2-methyl-1-butanol, and3-methyl-1-butanol. In a specific embodiment, the beneficial metaboliteis isobutanol.

In a method to produce a beneficial metabolite from a carbon source, theyeast microorganism is cultured in an appropriate culture mediumcontaining a carbon source. In certain embodiments, the method furtherincludes isolating the beneficial metabolite from the culture medium.For example, isobutanol may be isolated from the culture medium by anymethod known to those skilled in the art, such as distillation,pervaporation, or liquid-liquid extraction.

In one embodiment, the recombinant microorganism may produce thebeneficial metabolite from a carbon source at a yield of at least 5percent theoretical. In another embodiment, the microorganism mayproduce the beneficial metabolite from a carbon source at a yield of atleast about 10 percent, at least about 15 percent, about least about 20percent, at least about 25 percent, at least about 30 percent, at leastabout 35 percent, at least about 40 percent, at least about 45 percent,at least about 50 percent, at least about 55 percent, at least about 60percent, at least about 65 percent, at least about 70 percent, at leastabout 75 percent, at least about 80 percent, at least about 85 percent,at least about 90 percent, at least about 95 percent, or at least about97.5% theoretical. In a specific embodiment, the beneficial metaboliteis isobutanol.

This invention is further illustrated by the following examples thatshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing, areincorporated herein by reference for all purposes.

EXAMPLES General Methods for Examples 1-26

Sequences: Amino acid and nucleotide sequences disclosed herein areshown in Table 6.

TABLE 6 Amino Acid and Nucleotide Sequences of Enzymes and GenesDisclosed in Various Examples. Corresponding Protein Enz. Source Gene(SEQ ID NO) (SEQ ID NO) ALS B. subtilis Bs_alsS1_coSc (SEQ ID NO: 42)Bs_AlsS1 (SEQ ID NO: 43) KARI E. coli Ec_ilvC_coSc^(Q110V) (SEQ ID NO:44) Ec_IlvC^(Q110V) (SEQ ID NO: 45) E. coli Ec_ilvC_coSc^(P2D1-A1) (SEQID NO: 46) Ec_ilvC_coSc^(P2D1-A1) (SEQ ID NO: 47) KIVD L. lactisLl_kivD2_coEc (SEQ ID NO: 48) Ll_Kivd2 (SEQ ID NO: 49) DHAD L. lactisLl_ilvD_coSc (SEQ ID NO: 50) Ll_IlvD (SEQ ID NO: 51) S. cerevisiaeSc_ILV3ΔN (SEQ ID NO: 52) Sc_Ilv3ΔN (SEQ ID NO: 53) ADH D. Dm_ADH (SEQID NO: 54) Dm_Adh (SEQ ID NO: 55) melanogaster L. lactis Ll_adhA (SEQ IDNO: 56) Ll_AdhA (SEQ ID NO: 57) L. lactis Ll_adhA_coSc^(his6) (SEQ IDNO: 58) Ll_AdhA^(his6) (SEQ ID NO: 59) L. lactisLl_adhA^(RE1)_coSc^(his6) (SEQ ID NO: 60) Ll_AdhA^(RE1-his6) (SEQ ID NO:61)

Media: Medium used was standard yeast medium (see, for example Sambrook,J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3rd ed. 2001,Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press andGuthrie, C. and Fink, G. R. eds. Methods in Enzymology Part B: Guide toYeast Genetics and Molecular and Cell Biology 350:3-623 (2002)). YPmedium contains 1% (w/v) yeast extract, 2% (w/v) peptone. YPD is YPcontaining 2% glucose unless specified otherwise. YPE is YP containing25 mL/L ethanol. SC medium is 6.7 g/L Difco™ Yeast Nitrogen Base, 14 g/LSigma™ Synthetic Dropout Media supplement (includes amino acids andnutrients excluding histidine, tryptophan, uracil, and leucine), 0.076g/L histidine, 0.076 g/L tryptophan, 0.380 g/L leucine, and 0.076 g/Luracil. SCD is containing 2% (w/v) glucose unless otherwise noted.Drop-out versions of SC and SCD media are made by omitting one or moreof histidine (-H), tryptophan (-W), leucine (-L), or uracil (-U). Solidversions of the above described media contain 2% (w/v) agar.

Cloning techniques: Standard molecular biology methods for cloning andplasmid construction were generally used, unless otherwise noted(Sambrook, J., Russel, D. W. Molecular Cloning, A Laboratory Manual. 3ed. 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor LaboratoryPress). Cloning techniques included digestion with restriction enzymes,PCR to generate DNA fragments (KOD Hot Start Polymerase, Cat#71086,Merck, Darmstadt, Germany), ligations of two DNA fragments using the DNALigation Kit (Mighty Mix Cat# TAK 6023, Clontech Laboratories, Madison,Wis.), and bacterial transformations into competent E. coli cells(Xtreme Efficiency DH5a Competent Cells, Cat# ABP-CE-CC02096P, AlleleBiotechnology, San Diego, Calif.). Plasmid DNA was purified from E. colicells using the Qiagen QIAprep Spin Miniprep Kit (Cat#27106, Qiagen,Valencia, Calif.). DNA was purified from agarose gels using theZymoclean Gel DNA Recovery Kit (Zymo Research, Orange, Calif.; Catalog#D4002) according to manufacturer's protocols.

Colony PCR: Yeast colony PCR used the FailSafe™ PCR System (EPICENTRE®Biotechnologies, Madison, Wis.; Catalog #FS99250) according tomanufacturer's protocols. A PCR cocktail containing 15 μL of Master MixE buffer, 10.5 μL water, 2 μL of each primer at 10 μM concentration, 0.5μL polymerase enzyme mix from the kit was added to a 0.2 mL PCR tube foreach sample (30 μL each). For each candidate a small amount of cells wasadded to the reaction tube using a sterile pipette tip. Presence of thepositive PCR product was assessed using agarose gel electrophoresis.

SOE PCR: The PCR reactions were incubated in a thermocycler using thefollowing PCR conditions: 1 cycle of 94° C.×2 min, 35 cycles of 94°C.×30 s, 53° C.×30 s, 72° C.×2 min and 1 cycle of 72° C.×10 min. Amaster mix was made such that each reaction contained the following: 3μL MgSO₄ (25 mM), 5 μL 10×KOD buffer, 5 μL 50% DMSO, 5 μL dNTP mix (2 mMeach), 1 μL KOD, 28 μL dH₂O, 1.5 μL forward primer (10 μM), 1.5 μLreverse primer (10 μM), 0.5 μL template (plasmid or genomic DNA).

Genomic DNA Isolation: The Zymo Research ZR Fungal/Bacterial DNA Kit(Zymo Research Orange, Calif.; Catalog #D6005) was used for genomic DNAisolation according to manufacturer's protocols with the followingmodifications. Following resuspension of pellets, 200 μL was transferredto 2 separate ZR BashingBead™ Lysis Tubes (to maximize yield). Followinglysis by bead beating, 400 μL of supernatant from each of the ZRBashingBead™ Lysis Tubes was transferred to 2 separate Zymo-Spin™ IVSpin Filters and centrifuged at 7,000 rpm for 1 min. Following the spin,1.2 mL of Fungal/Bacterial DNA Binding Buffer was added to eachfiltrate. In 800 μl aliquots, filtrate from both filters was transferredto a single Zymo-Spin™ IIC Column in a collection tube and centrifugedat 10,000×g for 1 min. For the elution step, instead of eluting in 100μL of EB (elution buffer, Qiagen), 50 μL of EB was added, incubated 1min then the columns were centrifuged for 1 min. This elution step wasrepeated for a final elution volume of 100 μL.

S. cerevisiae Transformations. S. cerevisiae strains were grown in YPDcontaining 1% ethanol. Transformation-competent cells were prepared byresuspension of S. cerevisiae cells in 100 mM lithium acetate. Once thecells were prepared, a mixture of DNA (final volume of 15 μL withsterile water), 72 μL 50% PEG, 10 μL 1M lithium acetate, and 3 μL ofdenatured salmon sperm DNA (10 mg/mL) was prepared for eachtransformation. In a 1.5 mL tube, 15 μL of the cell suspension was addedto the DNA mixture (100 μL), and the transformation suspension wasvortexed for 5 short pulses. The transformation was incubated for 30 minat 30° C., followed by incubation for 22 min at 42° C. The cells werecollected by centrifugation (18,000×g, 10 seconds, 25° C.). The cellswere resuspended in 350 μL YPD and after an overnight recovery shakingat 30° C. and 250 rpm, the cells were spread over YPD plates containing0.2 g/L G418 selective plates. Transformants were then single colonypurified onto G418 selective plates.

K. marxianus transformations: K. marxianus strains were grown in 3 mL ofan appropriate culture medium at 250 rpm and 30° C. overnight. Thefollowing day, cultures were diluted in 50 mL of the same medium andgrown to an OD₆₀₀ of between 1 and 4. The cells were collected in asterile 50 mL conical tube by centrifugation (1600×g, 5 min at roomtemperature). The cells were resuspended in 10 mL of electroporationbuffer (10 mM Tris-C1, 270 mM sucrose, 1 mM MgCl₂, pH 7.5), andcollected at 1600×g for 5 min at room temperature. The cells wereresuspended in 10 mL IB (YPE, 25 mM DTT, 20 mM HEPES, pH 8.0; preparedfresh by diluting 100 μL of 2.5 M DTT and 200 μL of 1 M HEPES, pH 8.0into 10 mL of YPD). The cells were incubated for 30 min, 250 rpm, 30° C.(tube standing vertical). The cells were collected at 1600×g for 5 minat room temperature and resuspended in 10 mL of chilled electroporationbuffer. The cells were pelleted at 1600×g for 5 min at 4° C. The cellswere resuspended in 1 mL of chilled electroporation buffer andtransferred to a microfuge tube. The cells were collected bycentrifugation at >10,000×g for 20 sec at 4° C. The cells wereresuspended in appropriate amount of chilled electroporation buffer fora final biomass concentration of 30-38 OD₆₀₀/mL. 400 μL of cells wasadded to a chilled electroporation cuvette (0.4 cm gap), 50 μL of SOEPCR product (or water control) was added and mixed by pipetting up anddown, and the cuvette was incubated on ice for 30 min. The samples wereelectroporated at 1.8 kV, 1000 Ohm, 25 μF. The samples were thentransferred to a 50 mL tube with 1 mL of an appropriate culture medium,and the samples were incubated for overnight at 250 rpm at 30° C. Afterincubation the cells were plated onto appropriate agar plates.

K. lactis transformations: K. lactis strains were grown in 3 mL YPD at250 rpm and 30° C. overnight. The following day, cultures were dilutedin 50 mL YPD and allowed to grow until they reached an OD₆₀₀ of ˜0.8.Cells from 50 mL YPD cultures were collected by centrifugation (2700rcf, 2 min, 25° C.). The cells were washed with 50 mL sterile water andcollected by centrifugation at 2700 rcf for 2 min at RT. The cells werewashed again with 25 mL sterile water and collected by centrifugation at2700 rcf for 2 min at RT. The cells were resuspended in 1 mL 100 mMlithium acetate and transferred to a 1.5 mL Eppendorf tube. The cellswere collected by centrifugation for 10 sec at 18,000 rcf at RT. Thecells were resuspended in a volume of 100 mM lithium acetate that wasapproximately 4× the volume of the cell pellet. A volume of 10-15 μL ofDNA, 72 μL 50% PEG (3350), 10 μL 1 M lithium acetate, 3 μL denaturedsalmon sperm DNA, and sterile water were combined to a final volume of100 μL for each transformation. In a 1.5 mL tube, 15 μL of the cellsuspension was added to the DNA mixture and the transformationsuspension was vortexed with 5 short pulses. The transformation wasincubated for 30 min at 30° C., followed by incubation for 22 min at 42°C. The cells were collected by centrifugation for 10 sec at 18,000 rcfat RT. The cells were resuspended in 400 μL of an appropriate medium andspread over agar plates containing an appropriate medium to select fortransformed cells.

Analytical Chemistry:

Gas Chromatography (method GC1). Analysis of volatile organic compounds,including ethanol and isobutanol was performed on a Agilent5890/6890/7890 gas chromatograph fitted with an Agilent 7673Autosampler, a ZB-FFAP column (J&W; 30 m length, 0.32 mm ID, 0.25 μMfilm thickness) or equivalent connected to a flame ionization detector(FID). The temperature program was as follows: 200° C. for the injector,300° C. for the detector, 100° C. oven for 1 minute, 70° C./minutegradient to 230° C., and then hold for 2.5 min. Analysis was performedusing authentic standards (>99%, obtained from Sigma-Aldrich, and a5-point calibration curve with 1-pentanol as the internal standard.

High Performance Liquid Chromatography (method LC1): Analysis of organicacid metabolites including 2,3-dihydroxyisovalerate (DHIV),2,3-dihydroxy-2-methylbutanoic acid (DH2MB), isobutyrate and glucose wasperformed on an Agilent 1200 or equivalent High Performance LiquidChromatography system equipped with a Bio-Rad Micro-guard Cation HCartridge and two Phenomenex Rezex RFQ-Fast Fruit H+ (8%), 100×7.8-mmcolumns in series, or equivalent. Organic acid metabolites were detectedusing an Agilent 1100 or equivalent UV detector (210 nm) and arefractive index detector. The column temperature was 60° C. This methodwas isocratic with 0.0180 N H₂SO₄ in Milli-Q water as mobile phase. Flowwas set to 1.1 mL/min. Injection volume was 20 μL and run time was 16min. Quantitation of organic acid metabolites was performed using a5-point calibration curve with authentic standards (>99% or highestpurity available), with the exception of DHIV(2,3-dihydroxy-3-methyl-butanoate, CAS 1756-18-9), which was synthesizedaccording to Cioffi et al. (Cioffi, E. et al. Anal Biochem 1980, 104,pp. 485) and DH2MB which quantified based on the assumption that DHIVand DH2MB exhibit the same response factor. In this method, DHIV andDH2MB co-elute, hence their concentrations are reported as the sum ofthe two concentrations.

High Performance Liquid Chromatography (method LC4): Analysis of oxoacids, including 2,3-dihydroxyisovalerate (DHIV, CAS 1756-18-9),2,3-dihydroxy-2-methylbutyrate acid (DH2MB), lactate, acetate,acetolactate, isobutyrate, and pyruvate) was performed on a Agilent-1100High Performance Liquid Chromatography system equipped with an IonPacAS11-HC Analytical column (Dionex: 9 μm, 4.6×250 mm) coupled with anIonPac AG11-HC guard column (Dionex: 13 μm, 4.6×50 mm) and an IonPacATC-3 Anion Trap column (Dionex: 9×24 mm). Acetolactate was detectedusing a UV detector at 225 nm, while all other analytes were detectedusing a conductivity detector (ED50-suppressed conductivity with ASRS 4mm in AutoSuppression recycle mode, 200 mA suppressor current). Thecolumn temperature was 35° C. Injection size was 10 μL. This method usedthe following elution profile: 0.25 mM NaOH for 3 min, followed by alinear gradient from 0.25 to 5 mM NaOH in 22 min and a second lineargradient from 5 mM to 38.25 mM in 0.1 min, followed by 38.25 mM NaOH for4.9 min and a final linear gradient from 38.25 mM to 0.25 mM for 0.1 minbefore re-equilibrating at 0.25 mM NaOH for 7 min. Flow was set at 2mL/min. Analysis was performed using a 4-point calibration curve withauthentic standards (>99%, or highest purity available), with thefollowing exceptions: DHIV was synthesized according to Cioffi et al.(Cioffi, E. et al. Anal Biochem 1980, 104, pp. 485). DH2MB wassynthesized as described in Example 8 and quantified based on theassumption that DHIV and DH2MB exhibit the same response factor. Racemicacetolactate was made by hydrolysis ofEthyl-2-acetoxy-2-methylacetoacetate (EAMMA) with NaOH (Krampitz, L. O.Methods in Enzymology 1957, 3, 277-283.). In this method, DHIV and DH2MBare separated (FIG. 8).

Enzyme Assays

Determination of protein concentration: Protein concentration (of yeastlysate or of purified protein) was determined using the BioRad BradfordProtein Assay Reagent Kit (Cat#500-0006, BioRad Laboratories, Hercules,Calif.) and using BSA for the standard curve. A standard curve for theassay was made using a dilution series of a standard protein stock of500 μg/mL BSA. An appropriate dilution of cell lysate was made in waterto obtain OD₅₉₅ measurements of each lysate that fell within linearrange of the BioRad protein standard curve. Ten μL of the lysatedilution was added to 500 μL of diluted BioRad protein assay dye,samples were mixed by vortexing, and incubated at room temperature for 6min. Samples were transferred to cuvettes and read at 595 nm in aspectrophotometer. The linear regression of the standards was used tocalculate the protein concentration of each sample.

Alcohol Dehydrogenase (ADH) Assay. Cells were thawed on ice andresuspended in lysis buffer (100 mM Tris-HCl pH 7.5). 1000 μL of glassbeads (0.5 mm diameter) were added to a 1.5 mL Eppendorf tube and 875 μLof cell suspension was added. Yeast cells were lysed using a RetschMM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6×1 min each at fullspeed with 1 min incubations on ice between each bead-beating step. Thetubes were centrifuged for 10 min at 23,500×g at 4° C. and thesupernatant was removed for use. These lysates were held on ice untilassayed. Yeast lysate protein concentrations were determined asdescribed.

Dilutions of the samples were made such that an activity reading couldbe obtained. Generally the samples from strains expected to have low ADHactivity were diluted 1:5 in lysis buffer (100 mM Tris-HCl pH 7.5) andthe samples from strains with expected high ADH activity such as strainswhere the ADH gene is expressed from a high copy number plasmid werediluted 1:40 to 1:100. Reactions were performed in triplicate using 10μL of appropriately diluted cell extract with 90 μL of reaction buffer(100 mM Tris-HCl, pH 7.5; 150 μM NADH; 11 mM isobutyraldehyde) in a96-well plate in a SpectraMax® 340PC multi-plate reader (MolecularDevices, Sunnyvale, Calif.). The reaction was followed at 340 nm for 5minutes, with absorbance readings every 10 seconds. The reactions wereperformed at 30° C. The reactions were performed in complete buffer andalso in buffer with no substrate.

Isobutyraldehyde Oxidation Assay (ALD6 assay): Cell pellets were thawedon ice and resuspended in lysis buffer (10 mM sodium phosphate pH7.0, 1mM dithiothreitol, 5% w/v glycerol). One mL of glass beads (0.5 mmdiameter) was added to a 1.5 mL Eppendorf tube for each sample and 850μL of cell suspension were added. Yeast cells were lysed using a RetschMM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6×1 min each at fullspeed with 1 min incubation on ice between. The tubes were centrifugedfor 10 min at 21,500×g at 4° C. and the supernatant was transferred to afresh tube. Extracts were held on ice until assayed. Yeast lysateprotein concentrations were determined as described.

The method used to measure enzyme activity of enzymes catalyzing theoxidation of isobutyraldehyde to isobutyrate in cell lysates wasmodified from Meaden et al. 1997, Yeast 13: 1319-1327 and Postma et al.1988, Appl. Environ. Microbiol. 55: 468-477. Briefly, for each sample,10 μL of undiluted cell lysate was added to 6 wells of a UV microtiterplate. Three wells received 90 μL assay buffer containing 50 mMHEPES-NaOH at pH 7.5, 0.4 mM NADP⁺, 3.75 mM MgCl₂, and 0.1 mM, 1 mM, or10 mM isobutyraldehyde. The other 3 wells received 90 μL of no substratebuffer (same as assay buffer but without isobutyraldehyde). The bufferswere mixed with the lysate in the wells by pipetting up and down. Thereactions were then monitored at 340 nm for 5 minutes, with absorbancereadings taken every 10 seconds in a SpectraMax® 340PC plate reader(Molecular Devices, Sunnyvale, Calif.). The reactions were performed at30° C. The V_(max) for each sample was determined by subtracting thebackground reading of the no substrate control. A no lysate control wasalso performed in triplicate for each substrate concentration.

ALS Assay: For ALS assays described in Examples 1-18, cells were thawedon ice and resuspended in lysis buffer (50 mM potassium phosphate bufferpH 6.0 and 1 mM MgSO₄). 1000 μL of glass beads (0.5 mm diameter) wereadded to a 1.5 mL Eppendorf tube and 875 μL of cell suspension wasadded. Yeast cells were lysed using a Retsch MM301 mixer mill (RetschInc. Newtown, Pa.), mixing 6×1 min each at full speed with 1 minincubations on ice between each bead-beating step. The tubes werecentrifuged for 10 min at 23,500×g at 4° C. and the supernatant wasremoved for use. These lysates were held on ice until assayed. Proteincontent of the lysates was measured as described. All ALS assays wereperformed in triplicate for each lysate, both with and withoutsubstrate. To assay each lysate, 15 μL of lysate was mixed with 135 μLof buffer (50 mM potassium phosphate buffer pH 6.0, 1 mM MgSO₄, 1 mMthiamin-pyrophosphate, 110 mM pyruvate), and incubated for 15 minutes at30° C. Buffers were prepared at room temperature. A no substrate control(buffer without pyruvate) and a no lysate control (lysis buffer insteadof lysate) were also included. After incubation 21.5 μL of 35% H₂SO₄ wasadded to each reaction and incubated at 37° C. for 1 h.

For ALS assays described in Examples 19-25, cells were thawed on ice andresuspended in lysis buffer (100 mM NaPO₄ pH 7.0, 5 mM MgCl₂ and 1 mMDTT). One mL of glass beads (0.5 mm diameter) were added to a 1.5 mLEppendorf tube and 800 μL of the cell suspension was added to the tubecontaining glass beads. Yeast cells were lysed using a Retsch MM301mixer mill (Retsch Inc. Newtown, Pa.) and a cooling block by mixing sixtimes for 1 min each at 30 cycles/second with 1 min icing in betweenmixing. The tubes were centrifuged for 10 min at 21,500×g at 4° C. andthe supernatant was removed. Extracts were held on ice until assayed.Yeast lysate protein concentration was determined using the BioRadBradford Protein Assay Reagent Kit (Cat#500-0006, BioRad Laboratories,Hercules, Calif.) and using BSA for the standard curve as described. AllALS assays were performed in triplicate for each lysate. All buffers,lysates and reaction tubes were pre-cooled on ice. To assay each lysate,15 μL of lysate (diluted with lysis buffer as needed) was mixed with 135μL of assay buffer (50 mM KPi, pH 7.0, 1 mM MgSO₄, 1 mMthiamin-pyrophosphate, 110 mM pyruvate), and incubated for 15 min at 30°C. A no substrate control (buffer without pyruvate) and a no lysatecontrol (lysis buffer instead of lysate) were also included. Afterincubation each reaction was mixed with 21.5 μL of 35% H₂SO₄, incubatedat 37° C. for 1 h and centrifuged for 5 min at 5,000×g to remove anyinsoluble precipitants.

All assay samples were analyzed for the assay substrate (pyruvate) andproduct (acetoin) via high performance liquid chromatography an HP-1200High Performance Liquid Chromatography system equipped with two RestekRFQ 150×4.6 mm columns in series. Organic acid metabolites were detectedusing an HP-1100 UV detector (210 nm) and refractive index. The columntemperature was 60° C. This method was isocratic with 0.0180 N H₂SO₄ (inMilli-Q water) as mobile phase. Flow was set to 1.1 mL/min. Injectionvolume was 20 μL and run time was 8 min. Analysis was performed usingauthentic standards (>99%, obtained from Sigma-Aldrich) and a 5-pointcalibration curve.

TMA29 enzyme assay: Cell pellets were thawed on ice and resuspended inlysis buffer (10 mM sodium phosphate pH7.0, 1 mM dithiothreitol, 5% w/vglycerol). One mL of glass beads (0.5 mm diameter) was added to a 1.5 mLEppendorf tube for each sample and 850 μL of cell suspension were added.Yeast cells were lysed using a Retsch MM301 mixer mill (Retsch Inc.Newtown, Pa.), mixing 6×1 min each at full speed with 1 min incubationon ice between. The tubes were centrifuged for 10 min at 21,500×g at 4°C. and the supernatant was transferred to a fresh tube. Extracts wereheld on ice until assayed. Yeast lysate protein concentration wasdetermined using the BioRad Bradford Protein Assay Reagent Kit(Cat#500-0006, BioRad Laboratories, Hercules, Calif.) and using BSA forthe standard curve as described.

Enzymatic synthesis of (S)-2-acetolactate ((S)-AL) was performed in ananaerobic flask. The reaction was carried out in a total volume of 55 mLcontaining 20 mM potassium phosphate pH 7.0, 1 mM MgCl₂, 0.05 mMthiamine pyrophosphate (TPP), and 200 mM sodium pyruvate. The synthesiswas initiated by the addition of 65 units of purified B. subtilis AlsS,and the reaction was incubated at 30° C. in a static incubator for 7.5h.

Chemical synthesis of racemic 2-acetolactate ((R/S)-2-AL) was performedby mixing 50 μL of ethyl-2-acetoxy-2-methylacetoacetate (EAMMA) with 990μL of water. 260 μL of 2 N NaOH was then added in 10 μL increments with15 seconds of vortexing after each addition. The solution was then mixedon an orbital shaker for 20 minutes.

Chemical synthesis of racemic AHB ((R/S)-AHB) was performed by mixing 50μL of ethyl-2-acetoxy-2-ethyl-3-oxobutanoate with 990 μL of water. 2 NNaOH was then added in 10 μL increments with 15 seconds of vortexingafter each addition. The NaOH was added until the pH of the solution was12 (˜180 μL of 2 N NaOH). The solution was then mixed on an orbitalshaker for 20 minutes.

For determination of (S)-AL, (R/S)-AL or (R/S)-AHB reduction activity,10 μL of undiluted cell lysate was added to 6 wells of a UV microtiterplate. Three wells received 90 μL assay buffer containing 100 mM KPO₄ atpH 7.0, 150 μM NADPH, and 5 mM (S)-AL or 10 mM (R/S)-AL or 10 mM(R/S)-AHB as substrate. The other 3 wells received 90 μL of assay bufferbut without substrate. The buffers were mixed with the lysate in thewells by pipetting up and down. The reactions were then monitored at 340nm, with absorbance readings taken every 10 seconds in a SpectraMax®340PC plate reader (Molecular Devices, Sunnyvale, Calif.). The reactionswere performed at 30° C. The (S)-AL, (R/S)-AL or (R/S)-AHB reductionactivity for each sample was determined by subtracting the backgroundreading of the no substrate control. A no lysate control was alsoperformed in triplicate.

DHAD Enzyme Assay: Cell pellets were thawed on ice and resuspended inlysis buffer (50 mM Tris pH 8.0, 5 mM MgSO₄, and G BiosciencesYeast/Fungal ProteaseArrest™ (St. Louis, Mo., USA, Catalog #788-333)).One mL of glass beads (0.5 mm diameter) was added to a 1.5 mL Eppendorftube for each sample and 850 μL of cell suspension were added. Yeastcells were lysed using a Retsch MM301 mixer mill (Retsch Inc. Newtown,Pa.), mixing 6×1 min each at full speed with 1 min incubation on icebetween. The tubes were centrifuged for 10 min at 21,500×g at 4° C. andthe supernatant was transferred to a fresh tube. Extracts were held onice until assayed. Yeast lysate protein concentration was determined asdescribed. Protein from each sample was diluted in DHAD assay buffer (50mM Tris pH8, 5 mM MgSO₄) to a final concentration of 0.5 μg/μL. Threesamples of each lysate were assayed, along with no lysate controls. 10μL of each sample (or DHAD assay buffer) was added to 0.2 mL PCR tubes.Using a multi-channel pipette, 90 μL of the substrate was added to eachtube (substrate mix was prepared by adding 4 mL DHAD assay buffer to 0.5mL 100 mM DHIV). Samples were put in a thermocycler (EppendorfMastercycler) at 35° C. for 30 min followed by a 5 min incubation at 95°C. Samples were cooled to 4° C. on the thermocycler, then centrifuged at3000×g for 5 minutes. Finally, 75 μL of supernatant was transferred tonew PCR tubes and analyzed by HPLC as follows 100 μL DNPH reagent (12 mM2,4-Dinitrophenyl Hydrazine 10 mM Citric Acid pH 3.0 80% Acetonitrile20% MilliQ H₂0) was added to 100 μL of each sample. Samples wereincubated for 30 min at 70° C. in a thermo-cycler (Eppendorf,Mastercycler). Analysis of keto-isovalerate and isobutyraldehyde wasperformed on an HP-1200 High Performance Liquid Chromatography systemequipped with an Eclipse XDB C-18 reverse phase column (Agilent) and aC-18 reverse phase column guard (Phenomenex). Ketoisovalerate andisobutyraldehyde were detected using an HP-1100 UV detector (210 nm).The column temperature was 50° C. This method was isocratic with 70%acetonitrile to water as mobile phase with 2.5% dilute phosphoric acid(4%). Flow was set to 3 mL/min. Injection size was 10 μL and run time is2 min.

Example 1 Increased Isobutanol/Isobutyrate Ratio by Increasing ADHActivity in S. cerevisiae

The purpose of this example is to demonstrate that increased alcoholdehydrogenase activity results in an increased isobutanol yield, adecreased isobutyrate yield, and an increase in the ratio of isobutanolyield to isobutyrate yield.

Strains and plasmids disclosed in this example are shown in Tables 7 and8, respectively.

TABLE 7 Genotype of Strains Disclosed in Example 1. GEVO Number GenotypeGEVO2843 S. cerevisiae, MATa ura3 leu2 his3 trp1 pdc1Δ::P_(CUP1):[Bs_alsS1_coSc: T_(CYC1): P_(PGK1): Ll_kivD2: P_(ENO2): Sp_HIS5]pdc5Δ::[LEU2: bla: P_(TEF1): ILV3ΔN: P_(TDH3): Ec_ilvC_coSc^(Q110V)]pdc6Δ::[URA3: bla: P_(TEF1): Ll_kivD2: P_(TDH3): Dm_ADH] {evolved for C2supplement-independence, glucose tolerance and faster growth}

TABLE 8 Plasmids Disclosed in Example 1. Plasmid Name RelevantGenes/Usage Genotype pGV2011 2μ plasmid expressing P_(TDH3):Ec_ilvC_coSc^(Q110V), KARI, and DHAD P_(TEF1): Ll_ilvD_coSc, 2μ ori,bla, G418R pGV2485 2μ plasmid expressing P_(TDH3): Ec_ilvC_coSc^(Q110V),KARI, DHAD, and P_(TEF1): Ll_ilvD_coSc, ADH P_(ENO2): Ll_adhA, 2μ ori,bla, G418R

S. cerevisiae strain GEVO2843, which expresses a single alcoholdehydrogenase (D. melanogaster ADH, Dm_ADH) from its chromosomal DNA wastransformed with 2μ plasmids pGV2011 carrying only the KARI and DHAD(Ec_ilvC_Q110V and LI_ilvD_coSc, respectively) or pGV2485 carrying theKARI, DHAD and ADH (Ec_ilvC_Q110V, LI_ilvD_coSc, and LI_adhA,respectively) as described.

To start fermentation cultures, small overnight cultures of thetransformed strains were started in YPD medium containing 1% ethanol and0.2 g/L G418 and incubated overnight at 30° C. and 250 rpm. Threebiological replicates of each strain were tested. The next morning, theOD₆₀₀ of these cultures was determined and an appropriate amount used toinoculate 50 mL of the same medium in a 50 mL baffled flask to an OD₆₀₀of approximately 0.1. These precultures were incubated at 30° C. and 250rpm overnight. When the cultures had reached an OD₆₀₀ of approximately5-6 they were centrifuged at 2700 rpm for 5 min at 25° C. in 50 mLFalcon tubes. The cells from one 50 mL culture (one clone) wereresuspended in YPD containing 8% glucose, 0.2 g/L G418, 1% (v/v) ethanol(containing 3 g/L ergosterol and 132 g/L Tween-80), and buffered at pH6.5 with 200 mM MES. The cultures were then transferred into 250 mLunbaffled flasks and incubated at 30° C. and 75 rpm.

At the 72 h timepoint, samples from each fermentation flask were takenfor determining OD₆₀₀, ADH activity, and for analysis by GC1 and LC1. Toprepare samples for GC1 and LC1 analysis, an appropriate volume of cellculture was spun in a microcentrifuge for 10 minutes at maximum speedand the supernatant was removed for GC1 and LC1 analysis. Cell pelletswere prepared for ADH assays by centrifuging 14 mL of culture medium at3000×g for 5 minutes at 4° C. The supernatant was removed and the cellswashed in 3 mL cold, sterile water. The tubes were then centrifuged asper above for 2 minutes, the supernatant removed, and the tubesreweighed to determine total cell weight. The Falcon tubes were storedat −80° C. ADH assays were performed as described.

Table 9 shows the OD₆₀₀ for each strain during the course of thefermentation. During the 72 h of this fermentation, the OD₆₀₀ of thestrains were similar: they started at an OD₆₀₀ of around 7 and ended atan OD₆₀₀ of around 9. The in vitro ADH enzymatic activity of lysatesfrom GEVO2843 transformed with the two plasmids was measured for the 72h timepoint. Table 9 shows the ADH activity in the lysates as measuredin vitro. The strain carrying the plasmid with no ADH (pGV2011) showedan activity of about 0.04 U/mg. The strain carrying the plasmid with theLI_adhA gene, (pGV2485), had approximately 7-fold more ADH activity.

TABLE 9 OD₆₀₀ and Alcohol Dehydrogenase Activity of Strain GEVO2843Transformed with Plasmids pGV2011 or pGV2485 After 72 h of Fermentation.ADH activity GEVO2843 transformed with OD₆₀₀ [U/mg] pGV2011 8.5 0.04pGV2485 9.1 0.29

Isobutanol and isobutyrate titers after 72 h of fermentation are shownin Table 10. The isobutanol titer in the strain with low ADH activity of0.04 U/mg was significantly lower compared to the strain with high ADHactivity of 0.29 U/mg. The isobutyrate titer in the strain with low ADHactivity of 0.04 U/mg was significantly higher compared to the strainwith high ADH activity of 0.29 U/mg. Table 6 also shows the yield forisobutyrate and isobutanol after 72 h of fermentation. The isobutanolyield in the strain with low ADH activity of 0.04 U/mg was significantlylower compared to the strain with high ADH activity of 0.29 U/mg. Theisobutyrate yield in the strain with low ADH activity of 0.04 U/mg wassignificantly higher compared to the strain with high ADH activity of0.29 U/mg.

TABLE 10 Titers and Yields for Isobutanol and Isobutyrate in StrainGEVO2843 Transformed with Plasmids pGV2011 or pGV2485 After 72 h ofFermentation. Isobutanol Isobutyrate isobutanol yield Isobutryate yieldYield ratio titer titer [mol/mol [mol/mol (isobutanol/ [g/L] [g/L]glucose] glucose] isobutyrate) pGV2011 3.2 3.8 0.22 0.22 1.0 pGV2485 4.71.9 0.33 0.11 3.0

Example 2 Further Increased Isobutanol/Isobutyrate Ratio by Use ofVariant ADH LI AdhA^(RE1) in S. cerevisiae

The purpose of this example is to demonstrate that expression of analcohol dehydrogenase with increased k_(cat) and decreased K_(M) resultsin a further increase in isobutanol yield, decrease in isobutyrateyield, and increase in the ratio of isobutanol yield to isobutyrateyield.

TABLE 11 Genotype of Strains Disclosed in Example 2. GEVO NumberGenotype GEVO2843 S. cerevisiae, MATa ura3 leu2 his3 trp1pdc1Δ::P_(CUP1): [Bs_alsS1_coSc: T_(CYC1): P_(PGK1): Ll_kivD2: P_(ENO2):Sp_HIS5] pdc5Δ::[LEU2: bla: P_(TEF1): ILV3ΔN: P_(TDH3):Ec_ilvC_coSc^(Q110V)] pdc6Δ::[URA3: bla: P_(TEF1): Ll_kivD2: P_(TDH3):Dm_ADH] {evolved for C2 supplement-independence, glucose tolerance andfaster growth}

TABLE 12 Plasmids Disclosed in Example 2. Plasmid Name RelevantGenes/Usage Genotype pGV2543 2μ plasmid expressing P_(TDH3):Ec_ilvC_coSc^(Q110V), KARI, DHAD, KIVD, P_(TEF1): Ll_ilvD_coSc, and ADHP_(PGK1): Ll_kivD_coEc, (Ll_AdhA^(his6)) P_(ENO2): Ll_AdhA^(his6), 2μori, bla, G418R pGV2545 2μ plasmid expressing P_(TDH3):Ec_ilvC_coSc^(Q110V), KARI, DHAD, KIVD, P_(TEF1): Ll_ilvD_coSc, and ADHP_(PGK1): Ll_kivD coEc, (Ll_AdhA^(RE1-his6)) P_(ENO2):Ll_AdhA^(RE1-his6), 2μ ori, bla, G418R

S. cerevisiae strain GEVO2843, which expresses a single alcoholdehydrogenase (D. melanogaster ADH, Dm_ADH) from its chromosomal DNA wastransformed with 2p plasmids pGV2543 carrying KARI, DHAD, KIVD andhis-tagged, codon-optimized wild-type ADH (Ec_ilvC^(Q110V),LI_ilvD_coSc, and LI_adhA_coSc^(his6), respectively) or pGV2545 carryingKARI, DHAD, KIVD and his-tagged, codon-optimized mutant ADH(Ec_ilvC^(Q110V), LI_ilvD_coSc, and LI_adhA^(RE1)_coSc^(his6),respectively). These strains were cultured and evaluated for ADH enzymeactivity and the production of extracellular metabolites by GC1 and LC1as described.

The kinetic parameters of the gene products of LI_adhA_coSc^(his6)LI_adhA^(RE1)_coSc^(his6) (LI_adhA^(his6) and LI_adhA^(RE1-his6),respectively) are shown in Table 13.

TABLE 13 Comparison of Kinetic Parameters of Wild-Type Ll_adhA^(his6)with Modified Ll_adhA^(RE1) Measured for Isobutyraldehyde with NADH asCofactor. K_(M) k_(cat) k_(cat)/K_(M) Variant [mM isobutyraldehyde][s⁻¹] [M⁻¹*s⁻¹] Ll_adhA^(his6) 11.7 51 4400 Ll_adhA^(RE1-his6) 1.6 8449700

Table 14 shows the OD₆₀₀ for each strain during the course of thefermentation. During the 72 h of this fermentation, the OD₆₀₀ of thestrains were similar: they started at an OD₆₀₀ of around 6 and ended atan OD₆₀₀ of around 9. The in vitro ADH enzymatic activity of lysatesfrom GEVO2843 transformed with the two plasmids was measured for the 72h timepoint. Table 14 shows the ADH activity in the lysates as measuredin vitro as described above. The strain carrying the plasmid withLI_adhA_coSc^(his6) (pGV2543) showed an activity of about 0.38 U/mg. Thestrain carrying the plasmid with the LI_adhA^(RE1)_coSc^(his6) gene,(pGV2545), had approximately 7-fold more ADH activity.

TABLE 14 OD₆₀₀, and Alcohol Dehydrogenase Activity of Strain GEVO2843Transformed with Plasmids pGV2543 or pGV2545 After 72 h of Fermentation.ADH activity GEVO2843 transformed with OD₆₀₀ [U/mg] pGV2543 8.5 0.38pGV2545 8.8 2.46

Isobutanol and isobutyrate titers and yield after 72 h of fermentationare shown in Table 15. The isobutanol titer and yield in the straincarrying pGV2543 was lower compared to the strain carrying pGV2545. Theisobutyrate titer and yield in the strain carrying pGV2543 wassignificantly higher compared to the strain carrying pGV2545.

TABLE 15 Titers and Yields for Isobutanol and Isobutyrate in StrainGEVO2843 Transformed with Plasmids pGV2453 or pGV2485 After 72 h ofFermentation. GEVO2843 isobutanol yield Isobutryate yield Yield ratiotransformed Isobutanol Isobutyrate [mol/mol [mol/mol (isobutanol/ with[g/L] [g/L] glucose] glucose] isobutyrate) pGV2543 4.6 1.3 0.28 0.06 4pGV2545 4.9 0.3 0.29 0.01 20

Example 3 Further Increased Isobutanol/Isobutyrate Ratio in S.CEREVISIAE by Expression of RE1

The purpose of this example is to demonstrate that expression of analcohol dehydrogenase with increased k_(cat) and decreased K_(M) resultsin an increase in isobutanol yield and a decrease in isobutyrate yieldin fermentations performed in fermenter vessels.

A fermentation was performed to compare performance of S. cerevisiaestrains GEVO3519 and GEVO3523. Isobutanol and isobutyrate titers andyields were measured during the fermentation. GEVO3519 carries a 2pplasmid pGV2524 that contains genes encoding the following enzymes:KARI, DHAD, KIVD and his-tagged, codon-optimized wild-type Lactococcuslactis ADH. GEVO3523 carries a 2p plasmid pGV2524 that contains genesencoding the following enzymes: KARI, DHAD, KIVD and an improved variantof the his-tagged, codon-optimized Lactococcus lactis ADH havingdecreased K_(M) and increased k_(cat). These strains were evaluated forisobutanol, isobutyraldehyde, glucose consumption by LC1 and GC1, aswell as for OD₆₀₀ during a fermentation in DasGip fermenter vessels.

TABLE 16 Genotype of Strains Disclosed in Example 3. GEVO NumberGenotype GEVO3128 S. cerevisiae, MATa ura3 leu2 his3 trp1 gpd2Δ::[T_(KI)_(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)]gpd1Δ::P_(ccw12): hph pdc1Δ::[P_(CUP1): Bs_alsS1_coSc: T_(CYC1):P_(PGK1): Ll_kivDkivD2: P_(ENO2): Sp_HIS5] pdc5Δ::[LEU2: bla: P_(TEF1);ILV3ΔN: P_(TDH3): Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD:P_(TDH3): Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]{evolved for C2 supplement-independence, glucose tolerance and fastergrowth} GEVO3519 GEVO3128 transformed with plasmid pGV2524 GEVO3523GEVO3128 transformed with plasmid pGV2546

TABLE 17 Plasmids Disclosed in Example 3. Plasmid Name RelevantGenes/Usage Genotype pGV2524 2μ plasmid P_(TDH3):Ec_ilvC_coSc^(P2D1-A1), P_(TEF1): Ll_ilvD_coSc, P_(PGK1): Ll_kivD2_coEcP_(ENO2): Ll_adhA_coSc^(his6), 2μ ori, bla, G418R pGV2546 2μ plasmidP_(TDH3): Ec_ilvC_coSc^(P2D1-A1), P_(TEF1): Ll_ilvD_coSc, P_(PGK1):Ll_kivD2_coEc P_(ENO2): Ll_adhA_coSc^(RE1-his6), 2μ ori, bla, G418R

S. cerevisiae strain GEVO3128 was transformed with either 2μ plasmidpGV2524 or pGV2546, to generate strains GEVO3519 and GEVO3523,respectively as described. Inoculum cultures of GEVO3519 and GEVO3523were started by inoculating 500 mL baffled flasks containing 80 mL ofYPD medium 0.2 g/L G418 antibiotic, 1% v/v ethanol, and 0.019 g/Ltryptophan. The cultures were incubated for approximately 34 h. Theorbital shaker was set at 250 rpm and 30° C. in both experiments.Similar cell mass was achieved for GEVO3519 and GEVO3523 strains. Thecell density achieved after incubation was 8.0 OD₆₀₀. Batchfermentations were conducted in YPD medium containing 80 g/L glucose,0.2 g/L G418, 1% v/v ethanol, and 0.019 g/L tryptophan using 2 L topdrive motor DasGip vessels with a working volume of 0.9 L per vessel.Vessels were sterilized, along with the appropriate dissolved oxygen andpH probes, for 60 minutes at 121° C. Dissolved oxygen probes werecalibrated post sterilization in order to allow for polarization,however, pH probes were calibrated prior to sterilization. The pH wascontrolled at pH 6.0 using 6N KOH and 2N H₂SO₄. During the growth phaseof the culture the oxygen transfer rate (OTR) was 10 mM/h and during theproduction phase of the culture the OTR was 0.2 mM/h.

Table 18 shows the isobutanol titer and yield (as % theoretical) ascalculated for the production phase of the culture. Both isobutanoltiter and yield are increased in strain GEVO3523 carrying the alcoholdehydrogenase with decreased K_(M) and increased k_(cat). Table 18 alsoshows the isobutyrate titer, reported as maximum titer reached, andyield as carbon yield in %. Both isobutyrate titer and yield aredecreased in strain GEVO3523 carrying the alcohol dehydrogenase withdecreased K_(M) and increased k_(cat).

TABLE 18 Isobutanol and Isobutyrate Titers and Yields. IsobutanolIsobutyrate Isobutanol yield Isobutyrate yield Strain titer [g/L] [%theor.] titer [g/L] [% C-yield] GEVO3519 3.9 ± 0.4 50.5 ± 2.1 0.82 ±0.04 4.0 ± 0.0 GEVO3523 5.0 ± 0.3 59.5 ± 2.1 0.40 ± 0.01 2.0 ± 0.0

Example 4 Decreased Isobutyrate and Acetate Production in Fermentationswith Deletion of ALD6 Gene in S. cerevisiae

The following example illustrates that deletion of the ALD6 gene leadsto a decrease in isobutyrate and acetate production in fermentations.

Construction of ALD6 Deletion Strains: PCR was used to generate a DNAfragment that contained a deletion allele of ALD6 for deletion of ALD6from S. cerevisiae. One PCR reaction amplified a DNA fragment (A)comprising the upstream flanking region of ALD6 and a region of overlapat the 3′ end of the DNA fragment with the 5′ end of the P_(Sc) _(—)_(CCW12) promoter region from pGV1954, using primers oGV2834 andoGV2835. Another PCR reaction amplified a DNA fragment (D) comprisingthe downstream flanking region of ALD6 and a region of overlap at the 5′end of the DNA fragment with the 3′ end of the hph hygromycin resistanceORF from pGV2074, using primers oGV2836 and oGV2837. Another PCRreaction amplified a DNA fragment (B) comprising the P_(Sc) _(—)_(CCW12) promoter region from pGV1954 with a region of overlap at the 5′end of the DNA fragment with the 3′ end of the upstream flanking regionof ALD6 (fragment A) and a region at the 3′ end of the DNA fragment withthe 5′ end of the hph hygromycin resistance ORF from pGV2074, usingprimers oGV2631 and oGV2632. Another PCR reaction amplified a DNAfragment (C) comprising the hph hygromycin resistance ORF from pGV2074with a region of overlap at the 5′ end of the DNA fragment with the 3′end of the P_(Sc) _(—) _(CCW12) promoter region from pGV1954 (fragmentB) and a region of overlap at the 3′ end of the DNA fragment with the 5′end of the downstream flanking region of ALD6 (fragment D), usingprimers oGV2633 and oGV2634. DNA fragments A and B were combined by PCRusing primers oGV2834 and oGV2632 to generate DNA fragment AB and DNAfragments C and D were combined by PCR using primers oGV2633 and oGV2837to generate DNA fragment CD. DNA fragments AB and CD were combined byPCR using primers oGV2834 and oGV2837 to generate the final DNA fragmentABCD that contained the deletion allele of ALD6.

TABLE 19 Primer Sequences Disclosed in Example 4. oGV No. SequenceoGV968 ACTCGCCGATAGTGGAAACCGACG (SEQ ID NO: 62) oGV1965CAAACTGTGATGGACGACACC (SEQ ID NO: 63) oGV2631CAATACGTTATGCCGTAATGAAG (SEQ ID NO: 64) oGV2632GCTTTTTACCCATTATTGATATAGTGTTTAAGCGAATG (SEQ ID NO: 65) oGV2633CACTATATCAATAATGGGTAAAAAGCCTGAACTCAC (SEQ ID NO: 66) oGV2634TTATTCCTTTGCCCTCGGACG (SEQ ID NO: 67) oGV2680TGCACTGCTGTCTTCACTTC (SEQ ID NO: 68) oGV2796TGTCAGCGCTTCAGACTC (SEQ ID NO: 69) oGV2797AAGTATTTTTAAGGATTCGCTC (SEQ ID NO: 70) oGV2798CTTCATTACGGCATAACGTATTGAAGTATTTTTAAGGATTCGCTC (SEQ ID NO: 71) oGV2800CGTCCGAGGGCAAAGGAATAAGATAGTTATCATTATGTAAGTGCG (SEQ ID NO: 72) oGV2801GGGAGTTTAGCAATCAGC (SEQ ID NO: 73) oGV2802TGGTTGACCCGCAAACTTC (SEQ ID NO: 74) oGV2803ACAATCTCCCTGTCTCCTCCC (SEQ ID NO: 75) oGV2804AAGGTGATTTGGCACAAATTTTAC (SEQ ID NO: 76) oGV2805GGTACAATTCTGTCCTGAATTGTAG (SEQ ID NO: 77) oGV2806AGGTCCTAGAAATCCCTTAAG (SEQ ID NO: 78) oGV2808CTTCATTACGGCATAACGTATTGCGATATCAGTATACAAGGTAGGC (SEQ ID NO: 79) oGV2810CGTCCGAGGGCAAAGGAATAAGGATTTAAGATGAGTGGTATTGG (SEQ ID NO: 80) oGV2811TGTTCGTAACTTTTGTCATCAC (SEQ ID NO: 81) oGV2812TCAGCATGCGGAACAATTG (SEQ ID NO: 82) oGV2813TCCACACGGTATCATACGATC (SEQ ID NO: 83) oGV2814GCGGTCGACAAGTTCAATATG (SEQ ID NO: 84) oGV2815TACTGAGCCGCCAACCTTAGTA (SEQ ID NO: 85) oGV2816CATAACTATACCCGTACGCAG (SEQ ID NO: 86) oGV2818CTTCATTACGGCATAACGTATTGAGCGTAGATCTACTGAACATGC (SEQ ID NO: 87) oGV2820CGTCCGAGGGCAAAGGAATAACATGAGATTGTCAAAGAGG (SEQ ID NO: 88) oGV2821CACCAGGCTTATTGATGACC (SEQ ID NO: 89) oGV2822CATTACCGGCAGTTGCTC (SEQ ID NO: 90) oGV2824TATGACAGTGCCTATCAAGC (SEQ ID NO: 91) oGV2825AATGGGTTCTACCAGTATC (SEQ ID NO: 92) oGV2826AAGCCGGGAACGTGCGTAAC (SEQ ID NO: 93) oGV2827CTTCATTACGGCATAACGTATTGGGAACGCGTAATGGTGCTTG (SEQ ID NO: 94) oGV2828CGTCCGAGGGCAAAGGAATAACCCGAGTTGACTGCTCATTG (SEQ ID NO: 95) oGV2829AATACTCGCCGAGGCGTAGG (SEQ ID NO: 96) oGV2830TTGGAGCTGGGAGGTAAATC (SEQ ID NO: 97) oGV2831TGCGGCTAACCCATATTGAG (SEQ ID NO: 98) oGV2832TACGCTGAGCGTAGTACAAC (SEQ ID NO: 99) oGV2833TAAAGCGCTGGGTGGACAACCG (SEQ ID NO: 100) oGV2834GCACCGAGACGTCATTGTTG (SEQ ID NO: 101) oGV2835CTTCATTACGGCATAACGTATTGTAAACACGCCAGGCTTGACC (SEQ ID NO: 102) oGV2836CGTCCGAGGGCAAAGGAATAATCCATTCGGTGGTGTTAAGC (SEQ ID NO: 103) oGV2837ATGGCGAAATGGCAGTACTC (SEQ ID NO: 104) oGV2838ACCAACGACCCAAGAATC (SEQ ID NO: 105) oGV2839CTTTGCGACAGTGACAAC (SEQ ID NO: 106) oGV2840CCTCACGTAAGGGCATGATAG (SEQ ID NO: 107) oGV2841GCATTGCAGCGGTATTGTCAGG (SEQ ID NO: 108) oGV2842CAGCAGCCACATAGTATACC (SEQ ID NO: 109) oGV2843CTTCATTACGGCATAACGTATTGAGCCGTCGTTTGACATGTTG (SEQ ID NO: 110) oGV2844CGTCCGAGGGCAAAGGAATAACGCTCCATTTGGAGGGATCG (SEQ ID NO: 111) oGV2845GAATGCGCTTGCTGCTAGGG (SEQ ID NO: 112) oGV2846CAGCTCTTGCTGCAGGTAACAC (SEQ ID NO: 113) oGV2847GGCACAATCTTGGAGCCGTTAG (SEQ ID NO: 114) oGV2848ACCAAGCCATCAAGGTTGTC (SEQ ID NO: 115) oGV2849TGGGTGATGGTTTGGCGAATGC (SEQ ID NO: 116) oGV2896GAAATGATGACATGTGGAAATATAACAG (SEQ ID NO: 117)

Strains to demonstrate decreased isobutyrate and acetate production bydeletion of ALD6 were constructed by transformation of GEVO3198 with theABCD DNA fragment that contained the deletion allele of ALD6.Transformants were selected for resistance to 0.1 g/L hygromycin andtransformant colonies were screened by colony PCR for the correctintegration of the ABCD DNA fragment using primer pairs oGV2840/oGV2680,oGV968/oGV2841, and oGV2838/oGV2839. Strains GEVO3711, GEVO3712 andGEVO3713 were identified by this colony PCR as having ALD6 deleted bycorrect integration of the ABCD DNA fragment.

Strains containing an isobutanol production pathway to demonstratedecreased isobutyrate and acetate production by deletion of ALD6 wereconstructed by transformation of GEVO3711, GEVO3712 and GEVO3713 with a2p origin of replication plasmid, pGV2247, carrying genes expressingKARI, DHAD, KIVD and ADH (Ec_ilvC_coSc^(P2D1-A1), LI_ilvD_coSc,LI_kivD2_coEc, and LI_adhA, respectively). Transformants were selectedfor resistance to 0.2 g/L G418 and 0.1 g/L hygromycin and purified byre-streaking onto media containing 0.1 g/L hygromycin and 0.2 g/L G418,generating strains GEVO3714, GEVO3715 and GEVO3716. An ALD6 controlstrain containing an isobutanol production pathway, GEVO3466, wasgenerated by transformation of GEVO3198 with plasmid pGV2247.Transformants were selected for resistance to 0.2 g/L G418 and purifiedby re-streaking onto media containing 0.2 g/L G418.

Construction of ald2Δ, ald3Δ, ald4Δ, ald5Δ and hfd1Δ Deletion Strains:PCR was used to generate separate DNA fragments that containedindividual deletion alleles of ALD2, ALD3, ALD4, ALD5 and HFD1 fordeletion of ALD2, ALD3, ALD4, ALD5 and HFD1 individually from S.cerevisiae in separate strains. Additionally, PCR was used to generate aDNA fragment that contained a deletion allele covering both ALD2 andALD3, which are adjacent genes in the S. cerevisiae genome, for deletionof ALD2 and ALD3 together (ald2Δ ald3Δ) from S. cerevisiae in anindividual strain. Four-component fragments containing the upstreamflanking region, the P_(Sc) _(—) _(CCW12) promoter region from pGV1954,the hph hygromycin resistance ORF from pGV2074 and the downstreamflanking region for each individual gene were generated by PCR as forthe generation of the ABCD fragment for deletion of ALD6 except usingthe primer pairs listed in Table 20. The four-component fragment fordeletion of ALD2 and ALD3 together contained the upstream flankingregion from ALD2 and the downstream flanking region from ALD3 and wassimilarly constructed by PCR using the primer pairs listed in Table 20.The P_(Sc) _(—) _(CCW12) promoter region from pGV1954 was alwaysamplified with primer pair oGV2631/oGV2632 and the hph hygromycinresistance ORF from pGV2074 was always amplified with primer pairoGV2633/oGV2634.

TABLE 20 Primers Used to Amplify Upstream and Downstream Regions forGene Deletions. Primer Pairs for Primer Pairs for Gene Deletion UpstreamRegion Downstream Region ald2Δ oGV2796/oGV2797, oGV2800/oGV2801oGV2796/oGV2798 ald3Δ oGV2806/oGV2808 oGV2810/oGV2811 ald2Δ ald3ΔoGV2796/oGV2798 oGV2810/oGV2811 ald4Δ oGV2816/oGV2818 oGV2820/oGV2821ald5Δ oGV2826/oGV2827 oGV2828/oGV2829 ald6Δ oGV2834/oGV2835oGV2836/oGV2837 hfd1Δ oGV2842/oGV2843 oGV2844/oGV2845

Strains with deletion of ALD2, ALD3, ALD4, ALD5 and HFD1 individuallyand with deletion of ALD2 and ALD3 together were constructed bytransformation of GEVO3198 or GEVO3466 with the individualfour-component DNA fragment that contained the individual deletionallele of ALD2, ALD3, ALD4, ALD5 or HFD1 or with the four-component DNAfragment that contained the deletion allele of ALD2 and ALD3 together.Transformants were selected for resistance to 0.1 g/L hygromycin andtransformant colonies were screened by colony PCR for the correctintegration of the four-component DNA fragment using the primer pairslisted in Table 21. Strain GEVO3567 was identified by this colony PCR ashaving ALD2 correctly deleted; strain GEVO3568 was identified by thiscolony PCR as having ALD3 correctly deleted; strain GEVO3569 wasidentified by this colony PCR as having ALD2 and ALD3 together correctlydeleted; strain GEVO3579 was identified by this colony PCR as havingALD4 correctly deleted; strains GEVO3705, GEVO3706 and GEVO3707 wereidentified by this colony PCR as having ALD5 correctly deleted; andstrains GEVO3720, GEVO3721 and GEVO3722 were identified by this colonyPCR as having HFD1 correctly deleted.

Strains containing an isobutanol production pathway and with deletion ofALD2, ALD3 and ALD5 individually or with deletion of ALD2 and ALD3together were constructed by transformation of strains GEVO3567,GEVO3568, GEVO3569, GEVO3705, GEVO3706 and GEVO3707 with plasmidpGV2247. Transformants were selected for resistance to 0.2 g/L G418 and0.1 g/L hygromycin and purified by re-streaking onto media containing0.1 g/L hygromycin and 0.2 g/L G418, generating strains GEVO3586,GEVO3587, GEVO3588, GEVO3590, GEVO3591, GEVO3592, GEVO3593, GEVO3594,GEVO3595, GEVO3708, GEVO3709 and GEVO3710. Strains GEVO3579, GEVO3720,GEVO3721 and GEVO3722 were generated from GEVO3466 and thereforecontained plasmid pGV2247.

TABLE 21 Primers Used to Screen Colonies for Verification of GeneDeletions. Gene Dele- tion Primer Pairs ald2Δ oGV2802/oGV2632,oGV968/oGV2803, oGV2804/oGV2805 ald3Δ oGV2812/oGV2632, oGV968/oGV2813,oGV2814/oGV2815 ald2Δ oGV2802/oGV2632, oGV968/oGV2813, oGV2804/oGV2805,ald3Δ oGV2814/oGV2815 ald4Δ oGV2822/oGV2632, oGV968/oGV2896,oGV2824/oGV2825 ald5Δ oGV2832/oGV2680, oGV1965/oGV2833, oGV2830/oGV2831ald6Δ oGV2840/oGV2680, oGV968/oGV2841, oGV2838/oGV2839 hfd1ΔoGV2848/oGV2680, oGV968/oGV2849, oGV2846/oGV2847

TABLE 22 Genotype of Strains Disclosed in Example 4. GEVO No. GenotypeGEVO3198 MATa ura3 leu2 his3 trp1 gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2Δ::[T_(KI) _(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—)_(URA3)] pdc1Δ::[P_(CUP1): Bs_alsS_coSc: T_(CYC1): P_(PGK1):Ll_kivD2_coEc: P_(ENO2): Sp_HIS5] pdc5Δ::[LEU2: bla: P_(TEF1):Sc_ILV3ΔN: P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1):Ll_ilvD_coSc: P_(TDH3): Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA:P_(FBA1): Sc_TRP1] {evolved for C2 supplement-independence, glucosetolerance and faster growth} GEVO3466 MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—) _(URA3) _(—) _(short):P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] pdc1Δ::[P_(CUP1): Bs_alsS_coSc:T_(CYC1): P_(PGK1): Ll_kivD2_coEc: P_(ENO2): Sp_HIS5] pdc5Δ::[LEU2: bla:P_(TEF1): Sc_ILV3ΔN: P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1):Ll_ilvD_coSc: P_(TDH3): Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA:P_(FBA1): Sc_TRP1] {evolved for C2 supplement-independence, glucosetolerance and faster growth}: transformed with pGV2247 GEVO3567 MATaura3 leu2 his3 trp1 gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—)_(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)]pdc1Δ::[P_(CUP1): Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD2_coEc:P_(ENO2): Sp_HIS5] pdc5Δ::[LEU2: bla: P_(TEF1): Sc_ILV3ΔN:P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]ald2Δ::P_(Sc) _(—) _(CCW12): hph {evolved for C2supplement-independence, glucose tolerance and faster growth} GEVO3568MATa ura3 leu2 his3 trp1 gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI)_(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)]pdc1Δ::[P_(CUP1): Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD2_coEc:P_(ENO2): Sp_HIS5] pdc5Δ::[LEU2: bla: P_(TEF1): Sc_ILV3ΔN:P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]ald3Δ::P_(Sc) _(—) _(CCW12)-hph {evolved for C2 supplement-independence,glucose tolerance and faster growth} GEVO3569 MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—) _(URA3) _(—) _(short):P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] pdc1Δ::[P_(CUP1): Bs_alsS_coSc:T_(CYC1): P_(PGK1): Ll_kivD2_coEc: P_(ENO2): Sp_HIS5] pdc5Δ::[LEU2: bla:P_(TEF1): Sc_ILV3ΔN: P_(TDH3)-Ec-ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1):Ll_ilvD_coSc: P_(TDH3): Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA:P_(FBA1): Sc_TRP1] ald2Δ: ald3Δ::P_(Sc) _(—) _(CCW12): hph {evolved forC2 supplement-independence, glucose tolerance and faster growth}GEVO3579 MATa ura3 leu2 his3 trp1 gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2Δ::[T_(KI) _(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—)_(URA3)] pdc1Δ::[P_(CUP1): Bs_alsS_coSc: T_(CYC1): P_(PGK1):Ll_kivD2_coEc: P_(ENO2): Sp_HIS5] pdc5Δ::[LEU2: bla: P_(TEF1):Sc_ILV3ΔN: P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1):Ll_ilvD_coSc: P_(TDH3): Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA:P_(FBA1): Sc_TRP1] ald4Δ::P_(Sc) _(—) _(CCW12): hph {evolved for C2supplement-independence, glucose tolerance and faster growth};transformed with pGV2247 GEVO3586, MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—) _(URA3) _(—) _(short):P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] GEVO3587 and pdc1Δ::[P_(CUP1):Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD2_coEc: P_(ENO2): Sp_HIS5]GEVO3588 pdc5Δ::[LEU2: bla: P_(TEF1): Sc_ILV3ΔN:P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1] ald2Δ:ald3Δ::P_(Sc) _(—) _(CCW12): hph {evolved for C2supplement-independence, glucose tolerance and faster growth};transformed with pGV2247 GEVO3590, MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—) _(URA3) _(—) _(short):P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] GEVO3591 and pdc1Δ::[P_(CUP1):Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD2_coEc: P_(ENO2): Sp_HIS5]GEVO3592 pdc5Δ::[LEU2: bla: P_(TEF1): Sc_ILV3ΔN:P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]ald2Δ::P_(Sc) _(—) _(CCW12): hph {evolved for C2supplement-independence, glucose tolerance and faster growth};transformed with pGV2247 GEVO3593, MATa ura3 leu2 his3 trp1 gpd1::T_(KI)_(—) _(URA3) gpd2::T_(KI) _(—) _(URA3) _(—)_(short)-P_(FBA1)-KI_URA3-T_(KI) _(—) _(URA3) GEVO3594 andpdc1::P_(CUP1)-Bs_alsS_coSc-T_(CYC1)-P_(PGK1)-Ll_kivD2_coEc-P_(ENO2)-Sp_HIS5pdc5::LEU2-bla- GEVO3595P_(TEF1)-Sc_ILV3ΔN-P_(TDH3)-Ec_ilvC_coSc^(Q110V)pdc6::P_(TEF1)-Ll_ilvD_coSc-P_(TDH3)-Ec_ilvC_coSc^(P2D1-A1)-P_(ENO2)-Ll_adhA-P_(FBA1)-Sc_TRP1 ald3Δ::P_(Sc)_(—) _(CCW12)-hph {evolved for C2 supplement-independence, glucosetolerance and faster growth}; transformed with pGV2247 GEVO3705, MATaura3 leu2 his3 trp1 gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—)_(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] GEVO3706and pdc1Δ::[P_(CUP1): Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD2_coEc:P_(ENO2): Sp_HIS5] GEVO3707 pdc5Δ::[LEU2: bla: P_(TEF1): Sc_ILV3ΔN:P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]ald5Δ::P_(Sc) _(—) _(CCW12)-hph {evolved for C2 supplement-independence,glucose tolerance and faster growth} GEVO3708, MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—) _(URA3) _(—) _(short):P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] GEVO3709 and pdc1Δ::[P_(CUP1):Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD2_coEc: P_(ENO2): Sp_HIS5]GEVO3710 pdc5Δ::[LEU2: bla: P_(TEF1): Sc_ILV3ΔN:P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]ald5Δ::P_(Sc) _(—) _(CCW12): hph {evolved for C2supplement-independence, glucose tolerance and faster growth};transformed with pGV2247 GEVO3711, MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—) _(URA3) _(—) _(short):P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] GEVO3712 and pdc1Δ::[P_(CUP1):Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD2_coEc: P_(ENO2): Sp_HIS5]GEVO3713 pdc5Δ::[LEU2: bla: P_(TEF1): Sc_ILV3ΔN:P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]ald6Δ::P_(Sc) _(—) _(CCW12): hph {evolved for C2supplement-independence, glucose tolerance and faster growth} GEVO3714,MATa ura3 leu2 his3 trp1 gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI)_(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)]GEVO3715 and pdc1Δ::[P_(CUP1): Bs_alsS_coSc: T_(CYC1): P_(PGK1):Ll_kivD2_coEc: P_(ENO2): Sp_HIS5] GEVO3716 pdc5Δ::[LEU2: bla: P_(TEF1):Sc_ILV3ΔN: P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1):Ll_ilvD_coSc: P_(TDH3): Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA:P_(FBA1): Sc_TRP1] ald6Δ::P_(Sc) _(—) _(CCW12): hph {evolved for C2supplement-independence, glucose tolerance and faster growth};transformed with pGV2247 GEVO3720, MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::[T_(KI) _(—) _(URA3) _(—) _(short):P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] GEVO3721 and pdc1Δ::[P_(CUP1):Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD2_coEc: P_(ENO2): Sp_HIS5]GEVO3722 pdc5Δ::[LEU2: bla: P_(TEF1): Sc_ILV3ΔN:P_(TDH3)-Ec_ilvC_coSc^(Q110V)] pdc6Δ::[P_(TEF1): Ll_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]hfd1Δ::P_(Sc) _(—) _(CCW12): hph {evolved for C2supplement-independence, glucose tolerance and faster growth}:transformed with pGV2247

TABLE 23 Plasmids Disclosed in Example 4. Plasmid Name Genotype pGV2247P_(TEF1): Ll_ilvD_coSc P_(TDH3): Ec_ilvcC_coSc^(P2D1-A1) P_(PGK1):Ll_kivD2_coEc P_(ENO2): Ll_adhA 2μ-ori, pUC-ori, bla, G418R.

Shake Flask Fermentations: Fermentations were performed to compare theperformance of GEVO3466 to strains containing the ald2Δ, ald3Δ, ald2Δald3Δ, ald4Δ, ald5Δ, hfd1Δ and ald6Δ deletion mutations. Yeast strainswere inoculated from cell patches or from purified single colonies fromYPD agar plates containing 0.2 g/L G418 into 3 mL of YPD containing 0.2g/L G418 and 1% v/v ethanol medium in 14 mL round-bottom snap-cap tubes.The cultures were incubated overnight up to 24 h shaking at an angle at250 rpm at 30° C. Separately for each strain, these overnight cultureswere used to inoculate 50 mL of YPD containing 0.2 g/L G418 and 1% v/vethanol medium in a 250 mL baffled flask with a sleeve closure to anOD₆₀₀ of 0.1. These flask cultures were incubated overnight up to 24 hshaking at 250 rpm at 30° C. The cells from these flask cultures wereharvested separately for each strain by centrifugation at 3000×g for 5minutes and each cell pellet resuspended separately in 5 mL of YPDcontaining 80 g/L glucose, 1% v/v stock solution of 3 g/L ergosterol and132 g/L Tween 80 dissolved in ethanol, 200 mM MES buffer, pH 6.5, and0.2 g/L G418 medium. Each cell suspension was used to inoculate 50 mL ofYPD containing 80 g/L glucose, 1% v/v stock solution of 3 g/L ergosteroland 132 g/L Tween 80 dissolved in ethanol, 200 mM MES buffer, pH 6.5,and 0.2 g/L G418 medium in a 250 mL non-baffled flask with a ventedscrew-cap to an OD₆₀₀ of approximately 5. These fermentations wereincubated shaking at 250 rpm at 30° C. Periodically, samples from eachshake flask fermentation were removed to measure OD₆₀₀ and to preparefor gas chromatography (GC1) analysis, for isobutanol and othermetabolites, and for high performance liquid chromatography (LC1)analysis for organic acids and glucose. Samples of 2 mL were removedinto a microcentrifuge tube and centrifuged in a microcentrifuge for 10min at maximum rpm. One mL of the supernatant was analysis ofextracellular metabolites by GC1 and LC1 as described.

Deletion of ALD6 decreased isobutyrate and acetate production in shakeflask fermentations: The 52 h shake flask fermentation results forGEVO3466 and the ald6Δ strains GEVO3714, GEVO3715 and GEVO3716 aresummarized in Table 24. The ald6Δ strains GEVO3714, GEVO3715 andGEVO3716 produced 71% less isobutyrate than the ALD6 strain GEVO3466.The ald6Δ strains GEVO3714, GEVO3715 and GEVO3716 also produced 86% lessacetate than the ALD6 strain GEVO3466. Isobutanol yield in the ald6Δstrains GEVO3714, GEVO3715 and GEVO3716 was not appreciably differentthan the ALD6 strain GEVO3466. Isobutanol titer in the ald6Δ strainsGEVO3714, GEVO3715 and GEVO3716 was 23% higher than the ALD6 strainGEVO3466.

TABLE 24 Shake Flask Fermentation Results Demonstrating DecreasedIsobutyrate and Acetate Production by Deletion of ALD6 IsobutanolIsobutanol Isobutyrate Acetate Titer Yield [% Produced Produced Strain[g/L] theoretical] [g/L] [g/L] GEVO3466 2.6 ± 0.1 44 ± 2 0.48 ± 0.060.59 ± 0.04 (ALD6) GEVO3714, 3.2 ± 0.2 42 ± 2 0.14 ± 0.06 0.08 ± 0.01GEVO3715 and GEVO3716 (ald6Δ)

The 72 h shake flask fermentation results for GEVO3466 and the ald2Δ,ald3Δ, ald2Δ, ald3Δ, ald4Δ, ald5Δ and hfd1Δ strains are summarized inTable 25 and Table 26. Strains with deletions in ALD3, ALD2 and ALD3together or ALD4 had no decrease in isobutyrate production compared withthe wild-type ALDH strain GEVO3466. Strains with deletions in ALD2, ALD5or HFD1 had no appreciable decrease in isobutyrate production comparedwith the wild-type ALDH strain GEVO3466. Strains with deletions of bothALD2 and ALD3 together produced 19% less acetate than the wild-type ALDHstrain GEVO3466 but strains with individual deletions of ALD2, ALD3,ALD4, ALD5 or HFD1 had no appreciable decrease in acetate productioncompared with the wild-type ALD strain GEVO3466.

TABLE 25 Shake Flask Fermentation Results Demonstrating No Decrease inIsobutyrate and Acetate production by Deletion of ALD2, ALD3, ALD4 orALD2 and ALD3 Together. Isobutanol Isobutanol Isobutyrate Acetate TiterYield Produced Produced Strain [g/L] [% theoretical] [g/L] [g/L]GEVO3466 5.1 ± 0.1 42 ± 2 1.24 ± 0.15 0.95 ± 0.07 (wild-type) GEVO3590,5.2 ± 0.2 45 ± 2 1.21 ± 0.06 0.85 ± 0.07 GEVO3591 and GEVO3592 (ald2Δ)GEVO3593, 5.5 ± 0.6 45 ± 6 1.34 ± 0.16 0.91 ± 0.07 GEVO3594 and GEVO3595(ald3Δ) GEVO3596, 6.8 ± 0.1 51 ± 1 1.41 ± 0.09 0.77 ± 0.08 GEVO3597 andGEVO3598 (ald2Δ ald3Δ) GEVO3579 5.6 ± 0.7 46 ± 6 1.34 ± 0.13 0.89 ± 0.15(ald4Δ)

TABLE 26 Shake Flask Fermentation Results Demonstrating No Decrease inIsobutyrate and Acetate Production by Deletion of ALD5 or HFD1.Isobutanol Isobutanol Isobutyrate Acetate Titer Yield Produced ProducedStrain [g/L] [% theoretical] [g/L] [g/L] GEVO3466 4.0 ± 0.4 44 ± 7  0.47± 0.04 0.75 ± 0.05 (wild-type) GEVO3708, 3.8 ± 0.8 46 ± 15 0.41 ± 0.040.64 ± 0.08 GEVO3709 and GEVO3710 (ald5Δ) GEVO3720, 4.4 ± 1.0 54 ± 140.40 ± 0.07 0.56 ± 0.18 GEVO3721 and GEVO3722 (hfd1Δ)

Fermentations in benchtop fermenters: Fermentations in benchtopfermenters were performed to compare the performance of GEVO3466 (ALD6)to GEVO3714 and GEVO3715 (ald6Δ). Glucose consumption, isobutanolproduction, isobutyrate production, and OD₆₀₀ were measured during thefermentation. For these fermentations, purified strains from streakplates were transferred to 500 mL baffled flasks containing 80 mL of YPDmedium containing 1% v/v ethanol, 100 μM CuSO₄.5H₂0 and 0.2 g/L G418 andincubated for 32 h at 30° C. in an orbital shaker at 250 rpm. The flaskcultures were transferred to individual 2 L top drive motor fermentervessels with a working volume of 0.9 L of YPD medium containing 80 g/Lglucose, 1% v/v ethanol, 100 μM CuSO₄.5H₂0 and 0.2 g/L G418 per vesselfor a starting OD₆₀₀ of 0.5. Fermenters were operated at 30° C. and pH6.0 controlled with 6N KOH and 2N H₂SO₄ in a 2-phase aerobic conditionbased on oxygen transfer rate (OTR). Initially, fermenters were operatedat a growth phase OTR of 10 mM/h by fixed agitation of 700 rpm and anair overlay of 5 sL/h. Cultures were grown for 24 h to approximately9-10 OD₆₀₀ then immediately switched to a production aeration OTR=2.0mM/h by reducing agitation from 700 rpm to 450 rpm for the period of 24h to 86.5 h. Periodically, samples from each fermenter were removed tomeasure OD₆₀₀ and to prepare for gas chromatography (GC1) analysis, forisobutanol and other metabolites, and for high performance liquidchromatography (LC1) analysis for organic acids and glucose. Samples of2 mL were removed into a microcentrifuge tube and centrifuged in amicrocentrifuge for 10 min at maximum rpm. One mL of the supernatant wassubmitted for GC1 and LC1 analysis as described.

Deletion of ALD6 decreased isobutyrate and acetate production andincreased isobutanol yield in benchtop fermenter fermentations: The 86.5h benchtop fermenter fermentation results are summarized in Table 27.The ald6Δ strains GEVO3714 and GEVO3715 produced 38% less isobutyratethan the ALD6 strain GEVO3466. The ald6Δ strains GEVO3714 and GEVO3715also produced 61% less acetate than the ALD6 strain GEVO3466. Isobutanolyield in the ald6Δ strains GEVO3714 and GEVO3715 was 25% higher than theALD6 strain GEVO3466. Isobutanol titer in the ald6Δ strains GEVO3714 andGEVO3715 was also 35% higher than the ALD6 strain GEVO3466.

TABLE 27 Benchtop Fermenter Fermentation Results Demonstrating DecreasedIsobutyrate and Acetate Production and Increased Isobutanol Yield byDeletion of ALD6. Isobutanol Isobutanol Isobutyrate Acetate Titer YieldProduced Produced Strain [g/L] [% theoretical] [g/L] [g/L] GEVO3466  8.2± 0.1 32 ± 1 2.1 ± 0.1 2.3 ± 0.3 (ALD6) GEVO3714 11.1 ± 0.1 40 ± 0 1.3 ±0.1 0.9 ± 0.1 and GEVO3715 (ald6Δ)

Example 5 Determination of ALD6 Activity in S. cerevisiae

The following example illustrates that the isobutyraldehyde oxidationactivity is significantly decreased in an ald6Δ strain.

TABLE 28 Genotype of Strains Disclosed in Example 5. GEVO # GenotypeSource GEVO3527 MATα his3Δ-1 leu2Δ ATCC# 201389 (BY4742) lys2Δ ura3ΔGEVO3940 MATα his3Δ-1 OpenBiosystems cat# YSC1054 leu2Δlys2Δ ura3Δ(Yeast MATalpha collection) ald6Δ::kan^(R)

Yeast strains GEVO3940 from which the ALD6 (YPL061W) gene was deletedand its parent GEVO3527 were each cultured in triplicate by inoculating3 mL of YPD medium in a 14 mL culture tube in triplicate for eachstrain. Cultures were started from patches on YPD agar plate forGEVO3527 and on YPD agar plates containing 0.2 g/L G418 plates forGEVO3940. The cultures were incubated overnight at 30° C. and 250 rpm.The next day, the OD₆₀₀ of the overnight cultures were measured and thevolume of each culture to inoculate a 50 mL culture to an OD₆₀₀ of 0.1was calculated. The calculated volume of each culture was used toinoculate 50 mL of YPD in a 250 mL baffled flask and the cultures wereincubated at 30° C. and 250 rpm. The cells were harvested during mid-logphase at ODs of 1.6-2.1 after 7 h of growth. The cultures weretransferred to pre-weighed 50 mL Falcon tubes and cells were collectedby centrifugation for 5 minutes at 3000×g. After removal of the medium,cells were washed with 10 mL MilliQ H₂0. After removal of the water, thecells were centrifuged again at 3000×g for 5 minutes and the remainingwater was carefully removed using a 1 mL pipette tip. The cell pelletswere weighed and then stored at −80° C. until they were lysed andassayed for isobutyraldehyde oxidation activity as described.

As shown in Table 29, the specific activity of S. cerevisiae ALD6 inGEVO3527 lysates for the oxidation of 10 mM isobutyraldehyde was 13.9mU/mg. The same strain with an ALD6 deletion had a specific activity of0.6 mU/mg which is 22-fold less. The specific activity of S. cerevisiaeALD6 in GEVO3527 lysates for the oxidation of 1.0 mM isobutyraldehydewas 17.6 mU/mg. The same strain with an ALD6 deletion had a specificactivity of 2.1 mU/mg which is 8-fold less. The specific activity of S.cerevisiae ALD6 in GEVO3527 lysates for the oxidation of 0.1 mMisobutyraldehyde was 6.7 mU/mg. The same strain with an ALD6 deletionhad a specific activity of 1.3 mU/mg which is 5-fold less. These datademonstrate that the endogenous ALD6 enzyme is responsible for theisobutyrate byproduct of the isobutanol pathway in S. cerevisiae

TABLE 29 Specific Isobutyraldehyde Oxidation Activities of StrainsGEVO3527 and GEVO3940 Using Various Isobutyraldehyde Concentrations.Specific Activities were Measured in Lysates From 3 Parallel Cultures ofGEVO3527 and GEVO3940. Shown are the Averages and Standard Deviations ofthe Activities Measured in the Biological Replicate Cultures. Activity[mU/mg total protein] measured with isobutyraldehyde 0.1 mM 1.0 mM 10 mMStrain Isobutyraldehyde Isobutyraldehyde Isobutyraldehyde GEVO3527 6.7 ±0.4 17.6 ± 1.2 13.9 ± 0.4 GEVO3940 1.3 ± 0.2  2.1 ± 0.2  0.6 ± 0.1

Example 6 Further Decreased Isobutyrate Production with Deletion of ALD6Gene and Overexpression of an Improved Alcohol Dehydrogenase in S.cerevisiae

The following example illustrates that the combination of an ALD6deletion and overexpression of an ADH with improved kinetic propertiesleads to a further decrease in isobutyrate production and to a furtherincrease in isobutanol production.

Isobutyrate is a byproduct of isobutyraldehyde metabolism in yeast andcan comprise a significant fraction of the carbon yield. The followingyeast strains were constructed: GEVO3466 was constructed by transformingstrain GEVO3198 with a 2p plasmid, pGV2247, carrying genes encoding thefollowing enzymes: KARI, DHAD, KIVD and wild-type ADH(Ec_ilvC_coSc^(P2D1-A1), LI_ilvD_coSc, LI_kivD2_coEc, and LI_adhA,respectively). GEVO3198 expresses a single copy of alcohol dehydrogenase(L. lactis ADH, LI_adhA) from its chromosomal DNA. The second strain, ofwhich biological replicates are termed GEVO3714 and GEVO3715, wasconstructed by transforming two independent strains, GEVO3711 andGEVO3712, with a 2p plasmid pGV2247 carrying genes encoding thefollowing enzymes: KARI, DHAD, KIVD and wild-type ADH(Ec_ilvC_coSc^(P2D1-A1), LI_ilvD_coSc, LI_kivD2_coEc, and LI_adhA,respectively). GEVO3711 and 3712 express a single alcohol dehydrogenase(L. lactis ADH, LI_adhA) and have the ALD6 gene deleted from thechromosomal DNA. A third strain, of which biological replicates aretermed GEVO3855 and GEVO3856, was constructed by transforming a strain,GEVO3711, with 2μ plasmid pGV2602 carrying genes encoding the followingenzymes: KARI, DHAD, KIVD and a mutant ADH (Ec_ilvC_coSc^(P2D1-A1-his6),LI_ilvD_coSc, LI_kivD2_coEc, and LI_adhA^(RE1), respectively).

TABLE 30 Genotype of Strains Disclosed in Example 6. GEVO No. GenotypeGEVO3198 MATa ura3 leu2 his3 trp1 gpd1Δ::T_(Kl) _(—) _(URA3)gpd2Δ::[T_(Kl) _(—) _(URA3) _(—) _(short):P_(FBA1):Kl_URA3:T_(Kl) _(—)_(URA3)]pdc1Δ::[P_(CUP1):Bs_alsS_coSc:T_(CYC1):P_(PGK1):Ll_kivDkivD:P_(ENO2):Sp_HIS5]pdc5Δ::[LEU2:bla:P_(TEF1):ILV3ΔN:P_(TDH3):Ec_ilvC_coSc^(Q110V)]pdc6Δ::[P_(TEF):Ll_ilvD:P_(TDH3):Ec_ilvC_coSc^(P2D1-A1):P_(ENO2):Ll_adhA:P_(FBA1):Sc_TRP1]{evolved for C2 supplement-independence, glucose tolerance and fastergrowth} GEVO3466 MATa ura3 leu2 his3 trp1 gpd1Δ::T_(Kl) _(—) _(URA3)gpd2Δ::[T_(Kl) _(—) _(URA3) _(—) _(short):P_(FBA1):Kl_URA3:T_(Kl) _(—)_(URA3)]pdc1Δ::[P_(CUP1):Bs_alsS_coSc:T_(CYC1):P_(PGK1):Ll_kivDkivD:P_(ENO2):Sp_HIS5]pdc5Δ::[LEU2:bla:P_(TEF1):ILV3ΔN:P_(TDH3):Ec_ilvC_coSc^(Q110V)]pdc6Δ::[P_(TEF):Ll_ilvD:P_(TDH3):Ec_ilvC_coSc^(P2D1-A1):P_(ENO2):Ll_adhA:P_(FBA1):Sc_TRP1]Transformed with pGV2247 {evolved for C2 supplement-independence,glucose tolerance and faster growth} GEVO3711, MATa ura3 leu2 his3 trp1gpd1Δ::T_(Kl) _(—) _(URA3) GEVO3712 gpd2Δ::[T_(Kl) _(—) _(URA3) _(—)_(short):P_(FBA1):Kl_URA3:T_(Kl) _(—) _(URA3)]pdc1Δ::[P_(CUP1):Bs_alsS_coSc:T_(CYC1):P_(PGK1):Ll_kivD:P_(ENO2):Sp_HIS5]pdc5Δ::[LEU2:bla:P_(TEF1):ILV3ΔN:P_(TDH3):Ec_ilvC_coSc^(Q110V)]pdc6Δ::[P_(TEF):Ll_ilvD:P_(TDH3):Ec_ilvC_coSc^(P2D1-A1):P_(ENO2):Ll_adhA:P_(FBA1):Sc_TRP1]ald6Δ::P_(CCW12): hph {evolved for C2 supplement-independence, glucosetolerance and faster growth} GEVO3714, MATa ura3 leu2 his3 trp1gpd1Δ::T_(Kl) _(—) _(URA3) GEVO3715 gpd2Δ::[T_(Kl) _(—) _(URA3) _(—)_(short):P_(FBA1):Kl_URA3:T_(Kl) _(—) _(URA3)]pdc1Δ::[P_(CUP1):Bs_alsS_coSc:T_(CYC1):P_(PGK1):Ll_kivD:P_(ENO2):Sp_HIS5]pdc5Δ::[LEU2:bla:P_(TEF1):ILV3ΔN:P_(TDH3):Ec_ilvC_coSc^(Q110V)]pdc6Δ::[P_(TEF):Ll_ilvD:P_(TDH3):Ec_ilvC_coSc^(P2D1-A1):P_(ENO2):Ll_adhA:P_(FBA1):Sc_TRP1]ald6Δ::P_(CCW12): hph Transformed with pGV2247 {evolved for C2supplement- independence, glucose tolerance and faster growth} GEVO3855,MATa ura3 leu2 his3 trp1 gpd1Δ::T_(Kl) _(—) _(URA3) GEVO3856gpd2Δ::[T_(Kl) _(—) _(URA3) _(—) _(short):P_(FBA1):Kl_URA3:T_(Kl) _(—)_(URA3)]pdc1Δ::[P_(CUP1):Bs_alsS_coSc:T_(CYC1):P_(PGK1):Ll_kivD:P_(ENO2):Sp_HIS5]pdc5Δ::[LEU2:bla:P_(TEF1):ILV3ΔN:P_(TDH3):Ec_ilvC_coSc^(Q110V)]pdc6Δ::[P_(TEF):Ll_ilvD:P_(TDH3):Ec_ilvC_coSc^(P2D1-A1):P_(ENO2):Ll_adhA:P_(FBA1):Sc_TRP1]ald6Δ::P_(CCW12): hph Transformed with pGV2602 {evolved for C2supplement- independence, glucose tolerance and faster growth}

TABLE 31 Plasmids Disclosed in Example 6. Plasmid Name Genotype pGV2247P_(TEF1):Ll_ilvD_coSc, P_(TDH3):Ec_ilvC_coSc^(P2D1-A1),P_(PGK1):Ll_kivD2_coEc, P_(ENO2): Ll_adhA. 2μ-ori, pUC-ori, bla, G418R.pGV2602 P_(TEF1):Ll_ilvD_coSc, P_(TDH3):Ec_ilvC_coSc^(P2D1-A1-his6),P_(PGK1):Ll_kivD2_coEc, P_(ENO2): Ll_adhA^(RE1). 2μ-ori, pUC-ori, bla,G418R.

Two different sets of fermentations were performed. Fermentation set Awas performed to compare the performance of GEVO3466 (LI_adhA) toGEVO3714-GEVO3715 (LI-adhA, ald6Δ). Fermentation set B was performed tocompare the performance of GEVO3714 (LI_adhA, ald6Δ) toGEVO3855-GEVO3856 (LI_adhA^(RE1), ald6Δ) respectively. Glucoseconsumption, isobutanol production, isobutyrate production, and OD₆₀₀were measured during the fermentation. For these fermentations, singleisolate cell colonies grown on YPD agar plates were transferred to 500mL baffled flasks containing 80 mL of YPD medium containing 1% v/vEthanol, 100 μM CuSO₄.5H₂0, and 0.2 g/L G418 and incubated for 32 h at30° C. in an orbital shaker at 250 rpm. The flask cultures weretransferred to individual 2 L top drive motor fermenter vessels with aworking volume of 0.9 L of YPD medium containing 80 g/L glucose, 1% v/vEthanol, 100 μM CuSO₄.5H₂0, and 0.2 g/L G418 per vessel for a startingOD₆₀₀ of 0.5. Fermenters were operated at 30° C. and pH 6.0 controlledwith 6N KOH in a 2-phase aerobic condition based on oxygen transfer rate(OTR). Initially, fermenters were operated at a growth phase OTR of 10mM/h by fixed agitation of 700 rpm and an air overlay of 5 sL/h in bothexperiments. Cultures were grown for 24 h to approximately 9-10 OD₆₀₀then immediately switched to production aeration conditions for 48.5 h.In the first experiment, an OTR of 2.5-3.0 mM/h was sustained byreducing agitation from 700 rpm to 425 rpm while in the secondexperiment, an OTR of 2.0-2.5 mM/h was sustained by reducing agitationfrom 700 rpm to 400 rpm. Periodically, samples from each fermenter wereremoved to measure OD₆₀₀ and to prepare for gas chromatography (GC1) andliquid chromatography (LC1) analysis. For GC1 and LC1 , 2 mL sample wasremoved into an Eppendorf tube and centrifuged in a microcentrifuge for10 min at maximum. One mL of the supernatant was analyzed by GC1(isobutanol, other metabolites) and one mL analyzed by high performanceliquid chromatography (LC1) for organic acids and glucose.

The 72.5 h data from two separate fermentation sets A and B aresummarized in Tables 32 and 33. Fermentation set A compared GEVO3466 (WTADH) to GEVO3714 and 3715 (WT ADH, ald6Δ) while the fermentation set Bcompared GEVO3714 (WT ADH, ald6Δ) to GEVO3855 and 3856 (LI_adhA^(RE1),ald6Δ)

The data referring to fermentation set A (Table 32) show that isobutanoltiter and theoretical yield in the strain carrying LI_adhA with the ALD6gene deletion was 1.4- and 1.3-fold higher, respectively, compared tothe strain carrying LI_adhA without the ALD6 gene deletion. The straincarrying LI_adhA without ALD6 gene deletion (GEVO3466) had anisobutyrate yield (gram isobutyrate produced/gram glucose consumed) of0.040 g/g while the strains carrying LI_adhA with the ALD6 gene deletion(GEVO3714, GEVO3715) had a lower isobutyrate yield of 0.017 g/g. Thestrain carrying the L. lactis adhA without the ALD6 gene deletionproduced 2.3 g/L acetate while the strain carrying the L. lactis adhAwith the ALD6 gene deletion produced 0.6 g/L acetate.

TABLE 32 Data from Fermentation Set A. Isobutanol Isobutyrate IsobutanolIsobutyrate Acetate produced produced yield yield produced Strain OD₆₀₀[g/L] [g/L] [% theoretical] [g/g] [g/L] GEVO3466  9.7 ± 0.1  7.4 ± 0.61.7 ± 0.0 48.1 ± 2.6 0.040 ± 0.004 2.3 ± 0.1 (WT ADH) GEVO3714, 10.0 ±0.7 10.4 ± 0.1 0.8 ± 0.1 55.3 ± 0.6 0.017 ± 0.003 0.6 ± 0.1 GEVO3715 (WTADH, ALD6Δ)

The data referring to fermentation set B (Table 33) show that isobutanoltiter and theoretical yield in the strain carrying L. lactis adhA^(RE1)with the ALD6 gene deletion was 1.2 and 1.1-fold higher, respectively,compared to the strain carrying L. lactis adhA with the ALD6 genedeletion. The strains carrying L. lactis adhA^(RE1) with the ALD6 genedeletion (GEVO3855, GEVO3856) had the lowest isobutyrate yield (gramisobutyrate produced/gram glucose consumed), 0.005 g/g, and produced 0.0g/L acetate compared to the strain carrying L. lactis adhA with ALD6gene deletion (GEVO3714) which had a higher isobutyrate yield of 0.014g/g and a similar acetate titer of 0.0 g/L (Table 33).

TABLE 33 Data from Fermentation Set B. Isobutanol Isobutanol Isobutyrateyield Isobutyrate Acetate produced produced [% yield produced StrainOD₆₀₀ [g/L] [g/L] theoretical] [g/g] [g/L] GEVO3714, 9.7 ± 0.2 10.3 ±0.1 0.8 ± 0.0 46.5 ± 1.6 0.014 ± 0.000 0.0 ± 0.0 (WT ADH, ALD6Δ)GEVO3855, 9.9 ± 0.3 12.0 ± 0.0 0.3 ± 0.0 51.5 ± 0.8 0.005 ± 0.000 0.0 ±0.0 GEVO3856 (LI_adhA^(RE1), ALD6Δ)

Example 7 Identification of DH2MB as a by-Product of IsobutanolFermentation

During fermentation of isobutanol-producing yeast strains, it was foundthat an unknown peak, co-eluting with 2,3-dihydroxy isovalerate (DHIV)on method LC1, and quantitated on this basis, was acting as a sink for asubstantial portion of the carbon being utilized.

Initially, it was believed this peak was solely 2,3-dihydroxyisovalerate(DHIV), but subsequent studies indicated that KARI product inhibitionwould have occurred at these levels of DHIV, making such concentrationsimpossible. Additional experiments showed that this recovered peak wasnot reactive with DHAD in enzyme assays, thus eliminating thepossibility that significant amounts of DHIV were present.

High Performance Liquid Chromatography LC1: Analysis of organic acidmetabolites was performed on an Agilent-1200 High Performance LiquidChromatography system equipped with two Rezex RFQ-Fast Fruit H+ (8%)150×4.6 mm columns (Phenomenex) in series. Organic acid metabolites weredetected using an Agilent-1100 UV detector (210 nm) and refractive index(RI) detector. The column temperature was 60° C. This method wasisocratic with 0.0128 N H₂SO₄ (25% 0.0512 N H₂SO₄ in Milli-Q water) asmobile phase. Flow was set to 1.1 mL/min. Injection volume was 20 μL andrun time was 16 min.

High Performance Liquid Chromatography LC3: For samples containing amaximum of 10 mM aldehydes, ketones and ketoacid intermediates(combined), DNPH reagent was added to each sample in a 1:1 ratio. 100 μLDNPH reagent (12 mM 2,4-Dinitrophenyl Hydrazine 20 mM Citric Acid pH 3.080% Acetonitrile 20% MilliQ H₂O) was added to 100 μL of each sample.Samples were incubated for 30 min at 70° C. in a thermo-cycler(Eppendorf, Mastercycler). Analysis of acetoin, diacetyl,ketoisovalerate and isobutyraldehyde was performed on an Agilent-1200High Performance Liquid Chromatography system equipped with an EclipseXDB C-18 150×4 mm; 5 μm particle size reverse phase column (Agilent) anda C-18 reverse phase guard column (Phenomenex). All analytes weredetected using an Agilent-1100 UV detector (360 nm). The columntemperature was 50° C. This method was isocratic with 60% acetonitrile2.5% phosphoric acid (0.4%), 37.5% water as mobile phase. Flow was setto 2 mL/min. Injection size was 10 μL and run time is 10 min.

High Performance Liquid Chromatography LC4: Analysis of oxo acids wasperformed on a Agilent-1100 High Performance Liquid Chromatographysystem equipped with an IonPac AS11-HC Analytical, IonPac AG11-HC guardcolumn (3-4 mm for IonPac ATC column, Dionex) or equivalent and anIonPac ATC-1 Anion Trap column or equivalent. Oxo acids were detectedusing a conductivity detector (ED50-suppressed conductivity, Suppressortype: ASRS 4 mm in AutoSuppression recycle mode, Suppressor current: 300mA). The column temperature was 35° C. This method used the followingelution profile: Hold at 0.25 mM for 3 min; linear gradient to 5 mM at25 min; linear gradient to 38.5 mM at 25.1 min, hold at 38.5 mM for 4.9min; linear gradient to 0.25 mM at 30.1 min; hold for 7 min toequilibrate. Flow was set at 2 mL/min. Injection size is 5 μL and runtime is 37.1 min.

GC-MS: Varian 3800CP GC system equipped with a single quad 320MS; DB-5ms column; 1079 injection port at 250° C.; constant flow 1.0 mL/min at100 split ration; oven profile: initial temperature, 40° C., hold for 5min, ramp of 20° C./min up to 235° C. and hold for 2 minutes; combiPALautosampler delivering 0.5 μL of sample; collected masses of 35 to 100.BSTFA Derivation: (1) Evaporate sample to dryness under nitrogen in a GCvial; (2) add 0.5 mL of Acetonitrile and 0.5 mL of BSTFA reagent; (3)Incubate at 50° C. for 30 minutes; (4) Inject onto GC-MS.

LC-MS: For the LC-MS analysis of the LC1 peak fraction the sample wasinjected into an Agilent 1100 Series high-performance liquidchromatographic (HPLC) system that was equipped with a multiplewavelength detector and an LC/MSD Trap mass spectrometer (ion trap). Theseparations were monitored by mass spectrometry to provideidentification for the component in the sample. The mass spectrometerwas operated in the atmospheric pressure chemical ionization (APCI) modefor sample injection. The analyses were conducted using the positive andnegative APCI modes. Detection of the “unknown” was only observed in thenegative ionization mode. The analysis was conducted using MSn to obtainfragmentation data on the sample analyte. Separations were achievedusing a 4.6×150 mm Agilent Zorbax SB C-18 column with 5 μm particles.The sample was run using an isocratic method which used an eluent of 90%HPLC water and 10% methanol. A 10 μL injection was used for the analysisof the sample solution. The sample was also analyzed bypassing thechromatographic column.

DHIV and its isomer, DH2MB, elute at the same retention time on LC1. Thepeak related to these compounds is separated from other compounds in thefermentation samples. The peak was collected from the HPLC and used forfurther analysis.

The signal ratio of the RI detector signal to UV detector signal seen inLC1 for DHIV (and DH2MB) is characteristic of common organic acids (e.g.lactate, acetate, etc.); conjugated acids (e.g., pyruvate) have verydifferent RI/UV signal ratios. The recovered “peak DHIV” had thecharacteristics of a non-conjugated acid:

Ratio (RI/UV): Recovered DHIV/DH2MB peak (130); DHIV Std (150); Pyruvate(14).

The lack of a carbonyl moiety in the “mystery peak” was confirmed by thecomplete lack of reaction between the recovered peak fraction from LC1and DNPH: no adduct peaks were evident in the LC3 chromatographicsystem.

The recovered peak fraction from LC1 was then analyzed by method LC4,which runs under alkaline conditions, and is capable of separating DHIVand acetolactate. That result is shown in FIG. 9, together with anoverlay of standard mixtures. This clearly shows the separation betweenDH2MB (as it was subsequently identified), and DHIV. Some pyruvate wasalso brought along in the collection of the DH2MB peak.

NMR Analysis: The sample peak recovered from method LC1 was neutralizedand lyophilized and sent for NMR analysis. The 2-D connectivity analysisby 1H-COSY NMR (FIG. 10) and the proton NMR spectrum (FIG. 11) yieldedgood results.

2-D analysis of “mystery peak” eluting with DHIV (FIG. 10): One methylgroup, shifted downfield, is not split by any adjacent protons, wherethe methyl group at 0.95 ppm is split into a doublet by one protonadjacent to a hydroxyl. That proton, in turn, is split into a quartet bythe adjacent methyl group. Complex patterns between 3.1 and 3.7 ppmindicate the different anomers of glucose carried along during the peakcollection of “DHIV”.

The assignments of the NMR peaks are shown in the spectrum below (FIG.11), clearly indicating that the identity of the “mystery peak” is2,3-dihydroxy-2-butyrate (DH2MB).

The ¹H NMR and COSY spectra support the presence of2,3-dihydroxy-2-methylbutanoic acid, a structural isomer ofdihydroxyisovaleric acid. Other signals in these spectra support thepresence of anomeric proteins and, therefore, a sugar component.Furthermore, complex grouping of signals between 3.1-3.8 ppm are oftenobserved with oligosaccharides. The 13C NMR spectrum is very weak andappears to be an attached proton test (APT) experiment based on thesignal at 45 ppm that falls below the base line.

LC-MS was also carried out on the LC1 peak fraction. The LC-MS wassufficient to demonstrate that the compound had a mass of 134 (both DHIVand DH2MB) (FIG. 12).

This analysis conclusively identified the unknown by-product as2,3-dihydroxy-2-methylbutanoic acid (CAS #14868-24-7). This compoundexists in 4 different stereoisomeric forms.2,3-dihydroxy-2-methylbutanoic acid exists as a set of cis and transdiastereomers, each of which exists as a set of enantiomers. The fourcompounds are shown in FIG. 13.

As described herein, DH2MB is derived from(2S)-2-hydroxy-2-methyl-3-oxobutyrate (acetolactate). The product ofthis reaction would be either (2S,3R)-2,3-Dihydroxy-2-methylbutanoicacid, (2S,3S)-2,3-Dihydroxy-2-methylbutanoic acid or a mixture of thetwo diastereomers depending on the stereoselectivity of the endogenousenzyme(s) catalyzing this conversion.

Example 8 Production and Purification of DH2MB

The purpose of this example is to illustrate how DH2MB was produced andpurified.

An engineered S. cerevisiae CEN.PK2 strain comprising ALS activity(GEVO3160, S. cerevisiae CEN.PK2: MATa ura3 leu2 his3 trp1gpd1Δ::P_(CCW12): Hph gpd2Δ::T_(KI) _(—) _(URA3) _(—) _(short):P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3) pdc1Δ::P_(CUP1): Bs_alsS_coSc:T_(CYC1): P_(PGK1): LI_kivD: P_(ENO2) _(—) _(Sp) _(—) HIS5 pdc5Δ::LEU2:bla: P_(TEF1): ILV3ΔN: P_(TDH3): ilvC_coSc_Q110V pdc6Δ::P_(TEF1):LI_ilvD_P_(TDH3): Ec_ilvC_coSc_P2D1-A1: P_(ENO2): LI_adhA: P_(FBA-1):Sc_TRP1 {evolved for C2 supplement-independence, glucose tolerance andfaster growth} expressing plasmid pGV2247 (2-micron, G418 resistantplasmid for the expression of Ec_ilvC_P2D1-A1, LI_ilvD, LI_kivD2, andLI_adhA) was used to produce approximately 10 g/L DH2MB in a batchfermentation using a 2 L top drive motor DasGip vessels filled with 1 Lculture medium (10 g/L yeast extract, 20 g/L peptone, 80 g/L glucose, 1%v/v Ethanol, 100 μM CuSO₄.5H₂0, 0.2 g/L G418) at 30° C., pH6.0, and anOTR of approximately 10 mmol/h.

The cell-free fermentation broth was acidified to pH 2 usingconcentrated H₂SO₄. Acidified broth was concentrated to 350 mL underreduced pressure (0-100 mbar) using Büchi Rotovapor R-215. The flaskcontaining broth was heated in the water bath to 20-30° C. duringevaporation. A 70 mL volume of MeOH was added to concentrated broth andmixture was transferred to a 500 mL liquid-liquid extractor(Sigma-Aldrich cat. # Z562432), which was set up according tomanufacturer's specifications for continuous extraction with ethylacetate (EtOAc). Continuous extraction was carried out for 3 daysreplacing the EtOAc extract daily with fresh EtOAc.

Following extraction, the first two batches of DH2MB extract in EtOAcwere combined and dried with anhydrous MgSO₄ followed by filtration. Dryextract was concentrated under vacuum to 500 mL and was treated with 3 gof activated charcoal (Fluka cat#05105) for 30 min by stirring at roomtemperature. The decolorized solution was filtered and concentrated toapproximately 50 mL under vacuum (0-100 mbar using Büchi RotovaporR-215). The Solution was incubated at 4° C. for two days. Obtainedcrystals were filtered and washed with ice-cold diethylether andacetone. Crystals were dried using lyophilizer under reduced pressure(0.05 mbar) for one day.

Isolated DH2MB was analyzed by 1H (FIG. 14) and 13C (FIG. 15) NMR. ¹HNMR (TSP) 1.1 (d, 6.5 Hz, 3H), 1.3 (s, 3H), 3.9 (q, 6.5 Hz, 3H). A 13Cspectrum indicated five different carbon atoms present in the sample.Resonance at 181 ppm indicated carboxylic acid carbon present in thesample. In conclusion, based on NMR spectra one could estimate a 99%purity of isolated DH2MB.

Example 9 Impact of DH2MB Production on Isobutanol Yield in Fermentation

The purpose of this example is to demonstrate that DH2MB accumulates tosubstantial levels in yeast strains comprising ALS and TMA29 activity.

Strains and plasmids disclosed in this example are shown in Tables 34and 35, respectively.

TABLE 34 Genotype of S. cerevisiae Strain GEVO3160. Strain GenotypeGEVO3160 MATa ura3 leu2 his3 trp1 gpd1Δ::[P_(CCW12): hph] gpd2Δ::[T_(Kl)_(—) _(URA3) _(—) _(short): P_(FBA1): Kl_URA3:T_(Kl) _(—) _(URA3)]pdc1Δ:: [P_(CUP1): Bs_alsS_coSc: T_(CYC1): P_(PGK1): Ll_kivD: P_(ENO2):Sp_HIS5] pdc5Δ::[LEU2: bla: P_(TEF:)-ILV3ΔN: P_(TDH3):Ec_ilvC_coSc^(Q110V)] pdc6Δ:: [P_(TEF1): Ll_ilvD_P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]{evolvedfor C2 supplement-independence, glucose tolerance and faster growth}pGV2247

TABLE 35 Genotype of Plasmid pGV2247. Plasmid Genotype pGV2247 P_(Sc)_(—) _(TEF1): Ll_ilvD_coSc, P_(Sc) _(—) _(TDH3): Ec_ilvC_coSc^(P2D1A1),P_(Sc) _(—) _(TPI1): G418R, P_(Sc) _(—) _(PGK1): Ll_kivD_coEc, P_(Sc)_(—) _(ENO2): Ll_adhA, 2μ, AP^(r), PMB1

S. cerevisiae strain GEVO3160 was transformed with pGV2247 as described.A fermentation was performed to characterize the transformed strain. Asingle isolate cell colony grown on a YPD agar plate containing 0.2 g/LG418 were transferred 5 mL of YPD medium containing 80 g/L glucose, 1%v/v ethanol, 100 μM CuSO₄.5H₂0, and 0.2 g/L G418 and incubated for 24 hat 30° C., 250 rpm. Next, this culture was transferred to 500 mL baffledflasks containing 80 mL of the same medium and incubated for 24 h at 30°C. in an orbital shaker at 250 rpm. The flask culture was transferred toa 2 L top drive motor fermenter vessel with a working volume of 0.9 L ofthe same medium for a starting OD₆₀₀ of 0.5. The fermenter was operatedat 30° C. and pH 6.0 controlled with 6N KOH in a 2-phase aerobiccondition based on oxygen transfer rate (OTR). Initially, the fermenterwas operated at a growth phase OTR of 10 mM/h by fixed agitation of 700rpm and an air overlay of 5sL/h in both experiments. The cultures wasgrown for about 20 h to an OD₆₀₀ of approximately 8, and thenimmediately switched to production aeration. An OTR of 1 mM/h wassustained by reducing agitation from 700 rpm to 350 rpm. After 93 h postinoculation, one replicate vessel from each strain was further reducedto an OTR=0.3 mM/h by decreasing the agitation from 350 rpm to 180 rpm.Periodically, samples from each fermenter were removed to measure OD₆₀₀and to prepare for gas chromatography (GC1) and liquid chromatography(LC1) analysis. For GC1 and LC1, 2 mL sample was removed into anEppendorf tube and centrifuged in a microcentrifuge for 10 min atmaximum. One mL of the supernatant was analyzed by GC1 (isobutanol,other metabolites) and one mL analyzed by high performance liquidchromatography (LC1) for organic acids and glucose.

FIG. 16 depicts the product and by-product profiles of S. cerevisiaeGEVO3160 transformed with pGV2247. These profiles are representative forisobutanol producing Pdc-minus, Gpd-minus yeast strains.Pdc-minus/Gpd-minus yeast production strains are described in commonlyowned and co-pending publications, US 2009/0226991 and US 2011/0020889,both of which are herein incorporated by reference in their entiretiesfor all purposes. FIG. 16 shows that isobutanol (13.9 g/L) and theunknown compound quantified as “DHIV” and now identified as DH2MB (8.4g/L) are the primary products produced during microaerobic productionOTR. Assuming that the quantitation using the response factor of DHIVleads to an accurate quantitation of DH2MB, approximately 12-13% of thecarbon consumed is diverted into production of DH2MB. If theacetolactate that is converted into DH2MB would instead be convertedinto isobutanol then the isobutanol yield over the entire time of thefermentation shown in FIG. 16 would be significantly higher.

Example 10 ALS Expression is Necessary for DH2MB Production

The purpose of this example is to demonstrate that exogenously expressedALS activity is required for DH2MB accumulation in S. cerevisiae.

This experiment was performed to determine whether ALS is required forthe production of DH2MB. The strains used in this experiment wereGEVO1187 (S. cerevisiae CEN.PK2; MATa ura3-52 leu2-3_(—)112 his3Δ1trp1-289 ADE2) and GEVO2280 (S. cerevisiae CEN.PK2; MATa ura3 leu2 his3trp1 ADE2 pdc1Δ::P_(CUP1-1):Bs_alsS2:TRP1). Prior to fermentations, bothstrains were transformed with the 2 micron plasmid pGV2082(P_(TDH3):Ec_ilvC_coSc^(Q110V), P_(TEF1):LI_ilvD_coSc,P_(PGK1):LI_kivD_coEc, and P_(ENO2):Dm_ADH, 2μ ori, bla, G418R) asdescribed.

To measure ALS activity, yeast cell extracts from GEVO1187 and GEVO2280were prepared. Cells were grown to an OD₆₀₀ of about 1, induced with 1mM CuSO₄ for 2 hours and then harvested. To prepare cells for assays, 50ml of cells was collected by centrifugation at 2700×g. After removal ofthe media, cells were resuspended in sterile dH₂O, centrifuged at 2700×gand the remaining media was carefully removed with a 1 ml pipette tip.The cell pellets were weighed (empty tubes were preweighed) and thenfrozen at −80° C. until use. Cell lysates were made using the followingSOP as described below. Cells were thawed on ice and resuspended inlysis buffer (250 mM KPO₄ pH 7.5, 10 mM MgCl₂ and 1 mM DTT) such thatthe result was a 20% cell suspension by mass. A volume of 1000 μl ofglass beads (0.5 mm diameter) were added to a 1.5 ml Eppendorf tube and875 μl of cell suspension was added. Yeast cells were lysed using aRetsch MM301 mixer mill (Retsch Inc. Newtown, Pa.) by mixing 6×1 mineach at full speed with 1 min icing steps between. The tubes werecentrifuged for 10 min at 23,500×g at 4° C. and the supernatant wasremoved. Extracts were held on ice until assayed. The lysate proteinconcentration was determined using the BioRad Bradford Protein AssayReagent Kit (Cat#500-0006, BioRad Laboratories, Hercules, Calif.) andusing BSA for the standard curve as described. Briefly, all ALS assayswere performed in triplicate for each lysate, both with and withoutsubstrate. To assay each lysate, 100 μL of lysate diluted 1:2 with lysisbuffer was mixed with 900 μL of buffer (50 mM potassium phosphate bufferpH 6.0, 1 mM MgSO₄, 1 mM thiamin-pyrophosphate, 110 mM pyruvate), andincubated for 15 minutes at 30° C. Buffers were prepared at roomtemperature. A no substrate control (buffer without pyruvate) and a nolysate control (lysis buffer instead of lysate) were also included.After incubation 175 μL from each reaction was mixed with 25 μL 35%H₂SO₄ and incubated at 37° C. for 30 min. Samples were submitted toanalytics for analysis by LC1. Using this method, it was determined thatthe wild-type strain GEVO1187 had no detectable ALS activity while theALS-expressing strain GEVO2280 had 0.65 units/mg lysate ALS activity.

The performance of the two strains (with or without the heterologous ALSintegrated expression construct) was compared using the following shakeflask fermentation conditions. Strains were patched onto YPD platescontaining 0.2 mg/mL G418. After overnight growth, cells were removedfrom the plate with a sterile toothpick and resuspended in 4 mL of YPDwith 0.2 g/L G418. The OD₆₀₀ was determined for each culture. Cells wereadded to 50 mL YP with 50 g/L dextrose and 0.2 mg/mL G418 such that afinal OD₆₀₀ of 0.1 was obtained. To induce the CUP1 promoter driving ALSexpression, 1 mM copper sulfate was added at the 24 hour time point.Unused media was stored at 4° C. to act as medium blank for GC and LC,and to act as the t=0 sample for the fermentation. At t=24, 48 and 72hours samples were prepared for analysis by GC1 and at 72 hours sampleswere additionally analyzed by LC1. At 24 and 48 hours a 1:10 dilution ofthe supernatant of each culture was analyzed by YSI. If needed 50%glucose containing 0.2 g/L G418 was added to a final concentration of100 g/L glucose. Fermentations were performed at 30° C. shaking at 250RPM.

The DH2MB titer reached at 72 hours of a shake flask fermentation wasdetermined using LC1 method for both the WT strain (BUD1187) without ALSand the strain expressing the P_(CUP1):Bs_alsS2 at PDC1 (BUD2280). Eachstrain was transformed with the 4-component plasmid pGV2082. Thefermentation was performed as described. Without exogenous ALSexpression, the strain produced no DH2MB, whereas the strain with ALSexpression produced up to 1.4 g/L DH2MB plus DHIV.

Example 11 Only ALS Expression is Necessary for DH2MB Production

The purpose of this example is to demonstrate that ALS activity alone isresponsible for DH2MB accumulation in S. cerevisiae.

This experiment was performed to determine whether ALS alone or incombination with a KARI, DHAD, KIVD, ADH expressing plasmid isresponsible for the production of DH2MB. The strain used in thisexperiment was GEVO2618 (MATa ura3 leu2 his3 trp1 pdc1Δ::[P_(CUP1):Bs_alsS1_coSc: TRP1). The plasmids tested in this experiment werepGV2227 which contains the remaining four pathway genes(−P_(TEF1):LI_ilvD_coSc: P_(TDH3):Ec_ilvC_coSc^(Q110V):P_(Sc) _(—)_(TPI1): G418: P_(PGK1): LI_kivD2_coEc:PDC1-3′ region: P_(ENO2): LI_adhA2μ bla, pUC-ori), and pGV2020, the empty vector control (P_(Sc) _(—)_(TEF1), P_(Sc) _(—) _(TPI1), G418R, APr, 2μ).

Shake flask cultures of GEVO2618 transformed with pGV2020 and GEVO2618transformed with pGV2227 were started in YPD (15% glucose) containing200 mM MES pH6.5, and 0.4 g/L G418 at an OD600 ˜0.1, and were run at 30°C. and 75 rpm in a shaking incubator. Samples were taken at 24 h and 48h and the samples were analyzed for metabolite levels by HPLC (LC1) andGC (GC1). After 48 hours, all glucose was consumed from the media byboth strains. The strain containing the empty vector (GEVO2618+pGV2020)produced 4.6 g/L of DHIV+DH2MB representing 3.8% yield. The straincontaining the vector expressing additional four pathway genes(GEVO2618+pGV2227), produced a similar titer of 5.6 g/L DHIV+DH2MBrepresenting 3.1% yield.

Example 12 Effect of Increased KARI Activity on DH2MB Production

The purpose of this example is to demonstrate that increased KARIactivity results in decreased in DH2MB production in yeast comprisingALS activity.

Strains and plasmids disclosed in this example are shown in Tables 36and 37, respectively.

TABLE 36 Genotype of Strains Disclosed in Example 12. Strain GenotypeGEVO2843 S. cerevisiae, MATa ura3 leu2 his3 trp1pdc1Δ::P_(CUP1):[Bs_alsS1_coSc:T_(CYC1): P_(PGK1): Ll_kivD2: P_(ENO2):Sp_HIS5] pdc5Δ::[LEU2: bla: P_(TEF1): ILV3ΔN: P_(TDH3):Ec_ilvC_coSc^(Q110V)] pdc6Δ::[URA3: bla: P_(TEF1): Ll_kivD2: P_(TDH3):Dm_ADH] {evolved for C2 supplement-independence, glucose tolerance andfaster growth}

TABLE 37 Plasmids Disclosed in Example 12. Plasmid Genotype pGV2196 CEN,ARS, hph, bla, pUC-ori. pGV2377 P_(TEF1): Ll_ilvD_coSc, P_(ScPGK1):Ll_kivD_coEc, P_(ScENO2): Ll_adhA, 2μ ori, pUC ori, bla, G418R pGV2466P_(TEF1): Ll_ilvD_coSc, P_(SCTDH3): Ec_ilvC_coSc^(his6), P_(ScPGK1):Ll_kivD_coEc, P_(ScENO2): Ll_adhA, 2μ ori, pUC ori, bla, G418R pGV2398P_(TEF1): Ll_ilvD_coSc, P_(SCTDH3): Ec_ilvC_coSc^(Q110V-his6),P_(ScPGK1): Ll_kivD_coEc, P_(ScENO2):Ll_adhA, 2μ ori, pUC ori, bla,G418R pGV2400 P_(TEF1): Ll_ilvD_coSc, P_(SCTDH3):Ec_ilvC_coSc^(P2D1-A1-his6), P_(ScPGK1): Ll_kivD_coEc, P_(ScENO2):Ll_adhA, 2μ ori, pUC ori, bla, G418R pGV2406 P_(Sc) _(—) _(TEF1:)Ec_ilvC_coSc^(Q110V-his6), CEN, ARS, hph, bla, pUC ori.

S. cerevisiae strain GEVO2843 was transformed with 2μ plasmids pGV2377,pGV2466, pGV2398, and pGV2400 as described to determine if expression ofwild-type or engineered KARIs led to a greater accumulation of DH2MB.

Precultures of GEVO2843 transformed with the 2p plasmids (pGV2377, 2466,2398, 2400) were started in YPD containing 1% ethanol and 0.2 g/L G418and incubated overnight at 30° C. and 250 rpm. These precultures wereused to inoculate 50 mL of the same medium in a baffled flask andincubated at 30° C. and 250 rpm until reaching an OD₆₀₀ of ˜5. They werepelleted in 50 mL Falcon tubes at 2700 rcf for 5 minutes at 25° C. Next,the cells from each 50 mL culture were resuspended in 50 mL YPDcontaining 8% glucose, 1% (v/v) ethanol, ergosterol, Tween-80, 0.2 g/LG418, and 200 mM MES, pH6.5. The cultures were added to 250 mL unbaffledflasks and placed in an incubator at 30° C. and 75 rpm. Samples weretaken after 72 h to determine OD₆₀₀ and to analyze the fermentationbroth for extracellular metabolites via GC1 and LC1 analysis.

Table 38 shows that the strain transformed with pGV2377 (Notoverexpressing any KARI gene from plasmid) produced the highest carbonyield of 15% for combined DH2MB+DHIV, while the strains with pGV2466(containing Ec_ilvC_coSc^(his6)), pGV2398 (containingEc_ilvC_coSc^(Q110V-his6)) and pGV2400 (containingEc_ilvC_coSc^(P2D1-A1-his6)) had similar combined DH2MB+DHIV carbonyields of 8-10%. Likewise, the strain transformed with pGV2377 producedisobutanol at the lowest carbon yield of 6%. The remaining strainscomprising KARI genes on a plasmid produced isobutanol at higher carbonyields. The observation that decreased DH2MB production correlates withincreased isobutanol production is consistent with the finding thatDH2MB is produced from acetolactate via a reaction that does not involveKARI.

TABLE 38 Isobutanol and Combined DH2MB + DHIV Carbon Yields Isobutanolcarbon DH2MB + DHIV Strain Plasmid KARI yield [%] carbon yield [%]GEVO2843 pGV2377 n/a 6 15 GEVO2843 pGV2466 Ec_ilvC_coSc^(his6) 18 8GEVO2843 pGV2398 Ec_ilvC_coSc^(Q110V-his6) 15 8 GEVO2843 pGV2400Ec_ilvC_coSc^(P2D1-A1-his6) 18 10

A second experiment was performed in which strains expressed either noKARI from a plasmid, a low level of KARI, or a high level of KARI. Inthis experiment the KARI activity of cell lysates was measured.

S. cerevisiae strain GEVO2843 was transformed as described withcombinations of plasmids as described in Table 37; the no KARI straincontained pGV2377+pGV2196 and had no plasmid-borne KARI, the low KARIstrain contained pGV2377+pGV2406 and expressed KARI from a low copyplasmid, and the high KARI strain contained pGV2398+pGV2196 andexpressed KARI from a high copy plasmid. Fermentations and sampling wereperformed as described. GC1 and LC1 methods were performed as described.Cells for KARI assays were lysed as described except that lysis bufferwas 250 mM KPO₄ pH 7.5, 10 mM MgCl₂ and 1 mM DTT. The proteinconcentration of lysates was determined as described.

To measure in vitro KARI activity, acetolactate substrate was made bymixing 50 μl of ethyl-2 acetoxy-2-methyl-acetoacetate with 990 ul ofwater. Next 10 μl of 2 N NaOH was sequentially added, with vortex mixingbetween additions for 15 sec, until 260 μl of NaOH was added. Theacetolactate was agitated at room temperature for 20 min and held onice. NADPH was prepared in 0.01 N NaOH to a concentration of 50 mM. Theconcentration was determined by reading the OD of a diluted sample at340 nm in a spectrophotometer and using the molar extinction coefficientof 6.22 M⁻¹cm⁻¹ to calculate the precise concentration. Three bufferswere prepared and held on ice. Reaction buffer contained 250 mM KPO₄ pH7.5, 10 mM MgCl₂, 1 mM DTT, 10 mM acetolactate, and 0.2 mM NADPH. Nosubstrate buffer was missing the acetolactate. No NADPH buffer wasmissing the NADPH. Reactions were performed in triplicate using 10 μl ofcell extract with 90 μl of reaction buffer in a 96-well plate in aSpectraMax 340PC multi-plate reader (Molecular Devices, Sunnyvale,Calif.). The reaction was followed at 340 nm by measuring a kineticcurve for 5 minutes, with OD readings every 10 seconds at 30° C. TheVmax for each extract was determined after subtracting the backgroundreading of the no substrate control from the reading in complete buffer.

Table 39 shows data for KARI activity, as well as carbon yield in % forisobutanol and combined DH2MB+DHIV. As KARI activity increased theisobutanol carbon yield increased and the combined DH2MB+DHIV carbonyield decreased.

TABLE 39 KARI Activity, Isobutanol and Combined DH2MB + DHIV CarbonYields. DH2MB + Isobutanol DHIV KARI activity carbon carbon StrainPlasmid μmol/min/mg yield [%] yield [%] GEVO2843 pGV2377 + 0.011 ± .0025 19 pGV2196 GEVO2843 pGV2377 + 0.030* 11*  16* pGV2406 GEVO2843pGV2398 + 0.151 ± .005 19  11 pGV2196 *This data comprises only onesample

Example 13 Effect of Increased DHAD Activity

The purpose of this example is to demonstrate that increased DHADactivity results in decreased in DH2MB production in yeast comprisingALS activity.

Strains and plasmids disclosed in this example are shown in Tables 40and 41, respectively.

GEVO2843 was transformed with different pairs of plasmids. Strain Acontains pGV2227 plus pGV2196. Strain B contains pGV2284 plus pGV2196.Strain C contains pGV2284 plus pGV2336. Single transformants of BUD2843with one of the three 2-plasmid combinations were single colony purifiedon YPD plates containing hygromycin, and the patched cells were used toinoculate 3 mL YPD containing 1% ethanol (v/v), 0.2 g/L G418, and 0.1g/L hygromycin. The cultures were incubated at 30° C., 250 rpm overnightprior to their use to inoculate 3 mL YPD containing 1% ethanol (v/v),0.2 g/L G418, and 0.1 g/L hygromycin. These cultures were incubated at30° C., 250 rpm overnight. The following day, the cultures were used toinoculate 50 ml YPD containing 8% glucose, 200 mM MES pH6.5, Ergosterol,and Tween80 to an OD₆₀₀ of approximately 0.1. These cultures wereincubated at 30° C., 250 rpm overnight. The following day the cultureswere diluted in 50 mL of the same medium to an OD₆₀₀ of ˜0.1. Thecultures were incubated at 30° C., 250 rpm, and 1.5 mL samples wereremoved after 0, 24, 47, 70, and 92 hours of incubation. The sampleswere prepared for GC and LC analysis as described. After 92 hours, theremainder of all samples was centrifuged and the pellets were weighedand stored at −80° C. DHAD assays were performed with lysates preparedfrom the frozen pellets as described. LC1 and GC1 analysis was performedas described.

TABLE 40 Genotype of Strains Disclosed in Example 13. Strain GenotypeGEVO2843 MATa ura3 leu2 his3 trp1pdc1Δ::[P_(CUP1):Bs_alsS_coSc:T_(CYC1):P_(PGK1):Ll_kivD:P_(ENO2):Sp_HIS5]pdc5 Δ::[LEU2:bla:P_(TEF1):Sc_ILV3ΔN:P_(TDH3):Ec_ilvC_coSc^(Q110V)]pdc6Δ::[URA3:bla:P_(TEF1):Ll_kivD:P_(TDH3):DmADH] {evolved for C2supplement-independence, glucose tolerance and faster growth}

TABLE 41 Plasmids Disclosed in Example 13. Plasmids Genotype pGV2227P_(Sc) _(—) _(TEF1): Ll_ilvD_coSc, P_(Sc) _(—) _(TDH3):Ec_ilvC_coSc^(Q110V), P_(Sc) _(—) _(TPI1): G418, P_(Sc) _(—) _(PGK1):Ll_kivD_coEc, P_(Sc) _(—) _(ENO2): Ll_adhA, 2μ, AP^(r), PMB1 pGV2284P_(Sc) _(—) _(TEF1), P_(Sc) _(—) _(TDH3): Ec_ilvC_coSc^(P2D1-A1), P_(Sc)_(—) _(TPI1): G418, P_(Sc) _(—) _(PGK1): Ll_kivD_coEc, P_(Sc) _(—)_(ENO2): Ll_adhA, 2μ, AP^(r), PMB1 pGV2196 P_(Sc) _(—) _(PGK1) P_(Sc)_(—) _(TEF1), P_(Sc) _(—) _(TPI1): hph, CEN, AP^(r), pUC ORI pGV2336P_(Sc) _(—) _(ENO), T_(ScPDC6) P_(Sc) _(—) _(PGK), P_(Sc) _(—) _(TEF1):Ll_ilvD_coSc P_(Sc) _(—) _(TDH3), P_(Sc) _(—) _(TPI1): hph, CEN, AP^(r),pUC ORI

Table 42 shows the DHAD activity, isobutanol yield and the combinedDHIV+DH2MB yield. The strain transformed with pGV2284+pGV2196 (no DHADexpressed from a plasmid) produced the highest carbon yield of 19% forcombined DH2MB+DHIV and the lowest carbon yield of isobutanol at 9%. Thestrain transformed with pGV2227+pGV2196 (highest DHAD expression from aplasmid) had the lowest carbon yield of 9% for combined DH2MB+DHIV andthe highest carbon yield for isobutanol at 18%. The strain transformedwith pGV2284+pGV2336 (low copy DHAD expression from a plasmid) had anintermediate carbon yield of 16% for combined DH2MB+DHIV and of 12% forisobutanol.

TABLE 42 DHAD Activities, Isobutanol and Combined DH2MB + DHIV CarbonYields at 92 hrs Fermentation. DHAD Isobutanol carbon DH2MB + DHIVStrain Plasmids activity yield [%] carbon yield [%] A pGV2227 + 0.29 ±0.05 18 9 pGV2196 B pGV2284 + 0.05 ± 0.00 9 19 pGV2196 C pGV2284 + 0.08± 0.01 12 16 PGV2336

In a second experiment, GEVO2843 was transformed with different pairs ofplasmid (Table 43) and assessed in a shake flask fermentation as above.Strain D contains pGV2196 plus pGV2589. Strain E contains pGV2529 pluspGV2589. Strain F contains pGV2196 plus pGV2485. The strain transformedwith pGV2196+pGV2589 (no plasmid-borne DHAD) produced 1.25 g/Lisobutanol and 5.67 g/L DH2MB+DHIV. The strain with DHAD expressed froma high-copy plasmid (pGV2196+pGV2485) produced 2.74 g/L isobutanol and3.71 g/L DH2MB+DHIV, indicating that an increase in DHAD expression ledto a decrease in DH2MB+DHIV accumulation. The strain with DHAD expressedfrom a low-copy plasmid (pGV2529+pGV2485) produced an intermediate levelof both metabolites, consistent with an intermediate level of DHADactivity.

TABLE 43 Additional Plasmids Disclosed in Example 13. Plasmid Genotype2196 P_(Sc) _(—) _(PGK1), P_(Sc) _(—) _(TEF1), P_(Sc) _(—) _(TPI1)hph,CEN, AP^(r), pUC ORI 2529 P_(Sc) _(—) _(PGK1), P_(Sc) _(—)_(TEF1)Ll_ilvD_coSc4, P_(Sc) _(—) _(TPI1)hph, CEN, AP^(r), pUC ORI 2589P_(Sc) _(—) _(TDH3)Ec_ilvC_coSc_Q110V, P_(Sc) _(—) _(TPI1)G418R, P_(Sc)_(—) _(ENO2)Ll_adhA, 2μ, AP^(r), PMB1

TABLE 44 DHAD activities, Isobutanol Titer and Yield, and CombinedDH2MB + DHIV Titers at 72 hrs Fermentation. Plasmid-borne IsobutanolIsobutanol DH2MB + DHIV Strain Plasmid(s) DHAD Titer (g/L) Yield (%)(g/L) D pGV2196 + pGV2589 None 1.25 ± 0.27 16.1 5.67 ± 0.29 E pGV2529 +pGV2589 Low-copy 2.15 ± 0.05 24.8 5.00 ± 0.20 F pGV2196 + pGV2485High-copy 2.74 ± 0.22 31.0 3.71 ± 0.11

Example 14 Deletion of TMA29 in S. cerevisiae by Targeted Deletion

The following example illustrates that deletion of the TMA29 gene fromthe S. cerevisiae genome eliminates the production of DH2MB whenacetolactate synthase is overexpressed.

Several reductase enzyme candidates that may catalyze the production ofDH2MB were identified in the S. cerevisiae genome, including the TMA29gene product. The genes encoding these reductases were deleted in the S.cerevisiae strain GEVO2618, a strain known to produce g/L quantities ofDH2MB, using integration of a URA3 marker. Fermentations were performedwith these strains to determine if deleting any of the candidate genes,including TMA29, reduced or eliminated the production of DH2MB.

Strains, plasmids, and primer sequences are listed in Tables 45, 46, and47, respectively.

TABLE 45 Genotype of Strains Disclosed in Example 14. GEVO No. GenotypeGEVO1187 S. cerevisiae CEN.PK2 MATa ura3-52 leu2-3_112 his3Δ1 trp1-289ADE2 GEVO2618 S. cerevisiae, MATa ura3 leu2 his3 trp1pdc1Δ::[P_(CUP1-1): Bs_alsS1_coSc: TRP1]. GEVO3638 S. cerevisiae, MATaura3 leu2 his3 trp1 pdc1Δ::[P_(CUP1-1): Bs_alsS1_coSc: TRP1]tma29Δ::[T_(KI) _(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI)_(—) _(URA3]) GEVO3639 S. cerevisiae, MATa ura3 leu2 his3 trp1pdc1Δ::[P_(CUP1-1): Bs_alsS1_coSc: TRP1] tma29Δ::[T_(KI) _(—) _(URA3)_(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)] GEVO3640 S.cerevisiae, MATa ura3 leu2 his3 trp1 pdc1Δ::[P_(CUP1-1): Bs_alsS1_coSc:TRP1] tma29Δ::[T_(KI) _(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3:T_(KI) _(—) _(URA3)]

TABLE 46 Plasmids Disclosed in Example 14. Plasmid Name Genotype pGV1299KI_URA3, bla, pUC-ori. pGV2129 KI_URA3-5′, bla.

TABLE 47 Oligonucleotide Sequences Disclosed in Example 14. oGV #Sequence  893 GGATGTGAAGTCGTTGACACAG (SEQ ID NO: 118) 2231TTGAAACGTTGGGTCCATAC (SEQ ID NO: 119) 2232 TTCACCGTGTGCTAGAGAAC(SEQ ID NO: 120) 2862 TTATACAGGAAACTTAATAGAACAAATC (SEQ ID NO: 121) 2867TGAAACAGCATGGCGCATAG (SEQ ID NO: 122) 2869CTGTGTCAACGACTTCACATCCGAGGTAACGAGGAACAAGCC (SEQ ID NO: 123) 2870TTTCGCCGGTATATTCCGTAG (SEQ ID NO: 124) 2891GTTCTATTAAGTTTCCTGTATAACGGCATTGTTCACCAGAATGTC (SEQ ID NO: 125) 2902TCCCGACGGCTGCTAGAATG (SEQ ID NO: 126) 2904 CGCTCCCCATTAATTATACA(SEQ ID NO: 127) 2913 GAAAGGCTCTTGGCAGTGAC (SEQ ID NO: 128) 2914GCCCTGGTGCAATTAGAATG (SEQ ID NO: 129) 2915 TGCAGAGGGTGATGAGTAAG(SEQ ID NO: 130) 2916 GGCCAAAGGTAAGGAGAACG (SEQ ID NO: 131)

Strain Construction: S. cerevisiae strains GEVO3638, GEVO3639, andGEVO3640 were constructed by transforming GEVO2618 with bipartiteintegration SOE PCR products to replace TMA29 with a URA3 marker.Primers to amplify 5′ and 3′ targeting sequences for reductase geneswere designed with a 20 bp sequence homologous to a URA3 fragment. Thiswas done so that SOE PCR could be used to create fragments containingthe URA3 marker and homologous regions flanking the reductase gene ofinterest. PCR was performed on an Eppendorf Mastercycler® (Cat#71086,Novagen, Madison Wis.). The following PCR program was followed forprimer sets used to generate SOE PCR fragments: 94° C. for 2 min then 30cycles of (94° C. 30 sec, 53° C. 30 sec, 72° C. 1.5 min) then 72° C. for10 min. The following primer pairs and template were used for the firststep of the SOE reactions.

To generate the 5′ URA3 fragment, oGV2232 and oGV2862 were used toamplify the 5′ URA3 fragment using pGV2129 as template. The 1364 bpfragment was purified by gel electrophoresis. To generate the 3′ URA3fragment, oGV2231 and oGV893 were used to amplify the 3′ URA3 fragmentusing pGV1299 as template. The 1115 bp fragment was purified by gelelectrophoresis.

To generate the 5′ TMA29 fragment, oGV2867 and oGV2891 were used toamplify the 5′ TMA29 fragment using S. cerevisiae S288c genomic DNA astemplate. The S. cerevisiae S288c strain was purchased from ATCC(ATCC#204508). The 412 bp fragment was purified by gel electrophoresis.To generate the 3′ TMA29 fragment, oGV2869 and oGV2870 were used toamplify the 3′ TMA29 fragment using S. cerevisiae S288c genomic DNA astemplate. The 305 bp fragment was purified by gel electrophoresis.

The following primer pairs and templates were used to generate the SOEPCR products. To generate the 5′ TMA29 SOE PCR product, oGV2232 andoGV2867 were used. The 5′ URA3 fragment and the 5′ TMA29 fragment wereused as template. To generate the 3′ TMA29 SOE PCR product, oGV2231 andoGV2870 were used. The 3′ URA3 fragment and the 3′ TMA29 fragment wereused as template.

Transformation of S. cerevisiae strain GEVO2618 with the bipartiteintegration SOE PCR products was performed as described. Followingtransformation, the cells were collected by centrifugation (18,000×g, 10seconds, 25° C.) and resuspended in 400 μL SCD-HLWU media. Integrativetransformants were selected by plating the transformed cells on SCD-Uraagar medium. Once the transformants were single colony purified theywere maintained on SCD-Ura plates.

Colony PCR was used to verify correct integration. To screen for thecorrect 5′-end, the URA3: TMA29 5′ junction primers oGV2915 and oGV2902were used to give an expected band at 991 bp. To screen for the correct3′-end, the URA3: TMA29 3′ junction primers oGV2904 and oGV2916 wereused to give an expected band at 933 bp. To screen deletion of the TMA29gene primers oGV2913 and oGV2914 were used, expecting a lack of a 288 bpif the CDS was deleted.

Fermentations: Fermentations were conducted with tma29Δ strainsGEVO3638, GEVO3639, and GEVO3640 and the parent TMA29 strain GEVO2618.Cultures were started in YPD shaking at 30° C. and 250 rpm. After fourdoublings, the OD₆₀₀ was determined for each culture. Cells were addedto 50 mL YPD with 15% glucose such that a final OD₆₀₀ of 0.05 wasobtained. At t=24 h, 2 mL of media was removed and 25 μL used at a 1:40dilution to determine OD₆₀₀. The remaining culture was centrifuged in amicrocentrifuge at maximum speed for 10 min and 1 mL of supernatant wasremoved and submitted for LC1 and LC4 analysis. At t=48 h, 2 mL of mediawas removed and 25 μL used at a 1:40 dilution to determine OD₆₀₀. 1 mLof supernatant was submitted for LC1 analysis. In addition, 14 mL wascollected by centrifugation at 2700×g. After removal of the media, cellswere resuspended in sterile dH₂0, centrifuged at 2700×g and theremaining medium was carefully removed with a 1 mL pipette tip. The cellpellets were weighed (empty tubes were preweighed) and then frozen at−80° C. until thawed for ALS assays as described.

The production of DH2MB is dependent on heterologous ALS expression, forinstance the Bs_alsS1_coSc gene. The ALS activity of cell lysates wasmeasured as described to demonstrate that the TMA29 deletion had noimpact on ALS expression and/or activity. The ALS activity of extractsfrom the strains carrying the TMA29 deletion is not less than, and isslightly more than the activity of extracts from the parent strain. Theresults at 24 h (48 h for ALS activity) are summarized in Table 48 andclearly demonstrate the lack of DH2MB production in the strain with theTMA29 deletion. LC4 analysis confirmed that GEVO3527 did not produceDHIV.

TABLE 48 Production of DH2MB in Strain with TMA29 Deletion. GlucoseDH2MB by consumed by LC1 ALS activity Strain OD₆₀₀ LC1 [g/L] [g/L][U/mg] GEVO2618  9.2 ± 0.9 61.56 ± 12.0 1.51 ± 0.1 0.44 ± 0.06 GEVO3638,12.5 ± 5.0 68.44 ± 12.5 0.00 ± 0.0 0.57 ± 0.04 GEVO3639, GEVO3640(tma29Δ)

Example 15 Deletion of TMA29 in S. cerevisiae by Deletion Library

The following example illustrates that deletion of the TMA29 gene fromthe S. cerevisiae genome eliminates the production of DH2MB whenacetolactate synthase is overexpressed.

Strains, ORF deletions, and plasmids are listed in Tables 49, 50, and51.

TABLE 49 Genotype of Strains Disclosed in Example 15. GEVO #Genotype/Source GEVO3527 S. cerevisiae BY4742: MATa his3Δ1 leu2Δ0 lys2Δ0ura3Δ0/ATCC #201389, purchased from ATCC 10801 University BoulevardManassas, VA 20110-2209

TABLE 50 ORF Deletion Disclosed in Example 15. ORF deletion Gene nameSource YMR226C TMA29 Deletion library was obtained from Open Biosystems,cat # YSC 1054

TABLE 51 Plasmid Disclosed in Example 15. Plasmid Relevant Genes pGV2435P_(ScCUP1): Bs_alsS1_coSc: P_(ScTPI1): hph: T_(ScCYC1), CEN/ARS, bla,pUC-ori

A commercial library of S. cerevisiae strains which has one gene/ORFdeleted per strain was used to screen for a deletion that might catalyzethe production of DH2MB. The candidate strain containing the deletion ofthe TMA29 (i.e., YMR226C) ORF was selected. Since exogenous ALSexpression is required for production of DH2MB, a CEN plasmid (pGV2435)containing the Bs_alsS1_coSc gene driven by the CUP1 promoter wastransformed into the strains as described. Transformations wererecovered overnight at 30° C., 250 rpm before plating onto YPD platescontaining 0.2 g/L hygromycin. Transformants were then patched onto YPDplates containing 0.2 g/L hygromycin and incubated at 30° C.

Fermentations were performed with these strains to determine if deletingTMA29 (YMR226C) reduced or eliminated the production of DH2MB. Threeindependent transformants of each strain were used to inoculatefermentation precultures which were grown overnight to saturation in YPDcontaining 0.2 g/L hygromycin at 30° C. and 250 rpm. The next day, theOD₆₀₀ of the precultures was measured and the volume of overnightculture needed to inoculate a 50 mL culture to an OD₆₀₀ of 0.1 wascalculated for each culture. 50 mL of YPD containing 150 g/L glucose,200 mM MES, pH 6.5, and 0.2 g/L hygromycin in a 250 mL non-baffled flaskwere inoculated with the calculated amount of overnight culture. Cellswere incubated at 30° C. and 75 rpm in an orbital shaker. At 24 h, allcultures were fed an additional 75 g/L of glucose by addition of 8.8 mLof a 50% glucose solution to each flask and then returned to incubationat 30° C. and 75 rpm. At 72 h, 1.5 mL was sampled from each flask (750μL divided between two Eppendorf tubes). The OD₆₀₀ was measured for eachculture (1:40 dilution in H₂O). The cells were removed from samples bycentrifugation at ≧14000×g for 10 minutes in a microcentrifuge. Thesupernatants from the samples were collected and stored at 4° C. untilanalysis by LC1, and the cell pellets were stored at −80° C. untilthawed for ALS assays as described.

There was some variation in the growth between the two strains, withOD₆₀₀ values of 13.7 for GEVO3527 and 15.7 for the TMA29 deletion strainat 72 h (Table 52). The strains consumed the same amount of glucose ofaround 223 g/L by 72 h (Table 52). GEVO3527 produced 2.8 g/L of DH2MB by72 h. The YMR226C deletion strain (tma29Δ) did not produce detectablelevels of DH2MB. The specific DH2MB titer for GEVO3527 was 0.2 g/L/OD;the YMR226C deletion strain (tma29Δ) did not produce detectable levelsof DH2MB. LC4 analysis confirmed that GEVO3527 did not produce DHIV.

TABLE 52 Cell Growth, Glucose Consumed, and DH2MB Production at 72 h.Glucose DH2MB Specific consumed titer by DH2MB Strain OD₆₀₀ by LC1 [g/L]LC1 [g/L] titer [g/L/OD] GEVO3527 13.7 ± 0.3 223.3 ± 0.6 2.8 ± 0.1 0.2 ±0.01 TMA29Δ 15.7 ± 5.5 223.9 ± 0.2 0.0 ± 0.0 0.0 ± 0.0 

Example 16 Improved Isobutanol Rate, Yield, and Titer with Deletion ofTMA29 Gene in S. cerevisiae

The following example illustrates that deletion of the TMA29 gene fromthe S. cerevisiae genome leads to an increase in productivity, yield,and titer of the desired product, isobutanol. In addition, it leads to adecrease in DH2MB productivity, yield and titer.

DH2MB is a byproduct of acetolactate metabolism in yeast. In isobutanolfermentations, DH2MB can comprise 10% or greater of the carbon yield.Strains with wild-type TMA29 produce DH2MB in the presence of expressedacetolactate synthase (ALS), encoded by Bs_alsS1_coSc (SEQ ID NO: 23).Strains deleted for TMA29 do not produce DH2MB in the presence ofexpressed Bs_alsS1_coSc. A yeast strain deleted for all PDC and GPDgenes that expresses ALS (Bs_alsS1_coSc) from the chromosome was deletedfor TMA29 and transformed with a high copy four-component isobutanolpathway plasmid, pGV2550 with genes for DHAD (LI_ilvD_coSc), KARI(Ec_ilvC_coSc^(P2D1-A1-his6)), KIVD (LI_kivD2_coEc) and ADH(LI_adhA_coSc^(RE1-his6)). Isobutanol titer, yield and productivity ofthis strain were compared to that of the parent strain that was notdeleted for the TMA29 gene, in both a shake flask fermentation and infermenters. Strains and plasmids are listed in Tables 53 and 54,respectively.

TABLE 53 Genotype of Strains Disclosed in Example 16. GEVO No. GenotypeGEVO1187 S. cerevisiae CEN.PK2 MATa ura3 leu2 his3 trp1 ADE2 GEVO3351MATa ura3 leu2 his3 trp1 gpd1Δ::[TKI_URA3] gpd2Δ::TKI_URA3 pdc1Δ::[P_(CUP1): Bs_alsS1_coSc: T_(CYC1): P_(PGK1): Ll_kivD-P_(ENO2):Sp_HIS5] pdc5 Δ::[LEU2; bla; P_(TEF1): ILV3ΔN; P_(TDH3);ilvC_coSc^(Q110V)] pdc6 Δ::[P_(TEF)-Ll_ilvD: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1] {evolvedfor C2 supplement-independence, glucose tolerance and faster growth}GEVO3663 MATa ura3 leu2 his3 trp1 gpd1::T_(KI) _(—) _(URA3)gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::T_(KI) _(—) _(URA3) pdc1Δ::[P_(CUP1): Bs_alsS1_coSc: T_(CYC1): P_(PGK1): Ll_kivD-P_(ENO2):Sp_HIS5] pdc5 Δ::[LEU2; bla; P_(TEF1): ILV3ΔN; P_(TDH3);ilvC_coSc^(Q110V)] pdc6 Δ::[P_(TEF)-Ll_ilvD: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]tma29Δ::[T_(KI) _(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3: T _(KI)_(—) _(URA3)] {evolved for C2 supplement- independence, glucosetolerance and faster growth} GEVO3690, MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::T_(KI) _(—) _(URA3) GEVO3691, pdc1Δ::[P_(CUP1): Bs_alsS1_coSc: T_(CYC1): P_(PGK1): Ll_kivD-P_(ENO2):Sp_HIS5] GEVO 3692 pdc5 Δ::[LEU2; bla; P_(TEF1): ILV3ΔN; P_(TDH3);ilvC_coSc^(Q110V)] pdc6 Δ::[P_(TEF)-Ll_ilvD: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]Transformed with pGV2550 {evolved for C2 supplement-independence,glucose tolerance and faster growth} GEVO3694, MATa ura3 leu2 his3 trp1gpd1Δ::[T_(KI) _(—) _(URA3)] gpd2 Δ::T_(KI) _(—) _(URA3) GEVO3695, pdc1Δ::[P_(CUP1): Bs_alsS1_coSc: T_(CYC1): P_(PGK1): Ll_kivD-P_(ENO2):Sp_HIS5] GEVO3696 pdc5 Δ::[LEU2; bla; P_(TEF1): ILV3ΔN; P_(TDH3);ilvC_coSc^(Q110V)] GEVO3697 pdc6 Δ::[P_(TEF)-Ll_ilvD: P_(TDH3):Ec_ilvC_coSc^(P2D1-A1): P_(ENO2): Ll_adhA: P_(FBA1): Sc_TRP1]tma29Δ::[T_(KI) _(—) _(URA3) _(—) _(short): P_(FBA1): KI_URA3: T_(KI)_(—) _(URA3)]Transformed with pGV2550 {evolved for C2supplement-independence, glucose tolerance and faster growth}

TABLE 54 Plasmids Disclosed in Example 16. Plasmid Name Genotype pGV1299KI_URA3, bla, pUC-ori. pGV2129 KI_URA3-5′, bla, pUC ori pGV2550P_(ScTEF1): Ll_ilvD_coS, P_(ScTDH3): Ec_ilvC_coSc^(P2D1-A1-his6):P_(ScPGK1): Ll_kivD2_coEc: P_(ScENO2): Ll_adhA_coSc^(RE1-his6), 2μ-ori,pUC-ori, bla, G418R.

Yeast strain construction: GEVO3663 was constructed by transformingGEVO3351 with the bipartite integration SOE PCR products described inExample 14 to replace TMA29 with a URA3 marker as described, exceptafter transformation the cells were resuspended in 350 μL SCD-Ura mediabefore being spread to SCD-Ura plates.

S. cerevisiae strains GEVO3690, GEVO3691, and GEVO3692 were constructedby transforming GEVO3351 with plasmid pGV2550. S. cerevisiae strainsGEVO3694, GEVO3695, and GEVO3697 were constructed by transformingGEVO3663 with plasmid pGV2250 Briefly, competent cells were prepared byremoving cells from a fresh plate into 100 μL 100 mM lithium acetate.The cell suspension was incubated at room temperature for 30 min.Plasmid DNA was transformed as described. After transformation, thecells were resuspended in 400 μL YPD containing 1% ethanol and incubatedat 30° C. for 6 h shaking at 250 rpm. The cells were then spread ontoYPD plates containing 0.2 g/L G418. Transformants were single colonypurified onto YPD plates containing 0.2 g/L G418 plates. Once thetransformants were single colony purified they were maintained on YPDplates containing 0.2 g/L G418.

Fermentations: A shake flask fermentation was performed comparingperformance of GEVO3690-GEVO3692 (TMA29) to GEVO3694-GEVO3695 andGEVO3697 (tma294). Cultures (3 mL) were started in YPD containing 1%ethanol and 0.2 g/L G418 and incubated overnight at 30° C. and 250 rpm.The OD₆₀₀ of these cultures was measured after about 20 h. Anappropriate amount of each culture was used to inoculate 50 mL of YPDcontaining 1% ethanol and 0.2 g/L G418 in a 250 mL baffled flask to anOD₆₀₀ of approximately 0.1. These precultures were incubated at 30° C.and 250 rpm overnight. When the cultures had reached an OD₆₀₀ ofapproximately 5 they were centrifuged at 2700 rcf for 5 min at 25° C. in50 mL Falcon tubes. The cells from each 50 mL culture were resuspendedin 50 mL of fermentation media as described. The cultures were thentransferred to 250 mL unbaffled screw-cap flasks with small vents andincubated at 30° C. and 75 rpm. At 24 and 48 h, samples from each flaskwere removed to measure OD₆₀₀ and to prepare for GC1 analysis. For GC1,2 mL sample was removed into an Eppendorf tube and centrifuged in amicrocentrifuge for 10 min at maximum. One mL of the supernatant wasanalyzed by GC1. At 72 h the same procedures were used to collect cellsfor OD₆₀₀ and GC analysis and in addition the samples were analyzed byhigh performance liquid chromatography (LC1) for organic acids,including DH2MB and DHIV, and glucose.

The results at 72 h are summarized in Table 55. Isobutanol titer, yieldand rate increase with deletion of the TMA29 gene, while DH2MBproduction decreases.

TABLE 55 Isobutanol Titer, Yield, and Rate Increase at 72 h. GlucoseIsobutanol DH2MB consumed produced Isobutanol yield Isobutanol producedStrain OD₆₀₀ [g/L] [g/L] [% theoretical] rate [g/L/h] [g/L] GEVO3690,8.3 ± 0.3 29.8 ± 1.3 5.5 ± 0.4 45.1 ± 4 0.08 3.1 GEVO3691, GEVO3692GEVO3694, 8.3 ± 0.7 33.4 ± 1.0 7.6 ± 0.2 55.1 ± 2 0.11 0.03 GEVO3695,GEVO3697 (TMA29Δ)

In addition, the performance of GEVO3690-GEVO3691 (TMA29) toGEVO3694-GEVO3696 (tma29Δ) was also compared in fermentations performedin fermenter vessels. Plated cultures were transferred to 500 mL baffledflasks containing 80 mL of YP medium with 20 g/L glucose, 1% v/vEthanol, 100 μM CuSO₄.5H₂0, and 0.2 g/L G418 and incubated for 34.5 h at30° C. in an orbital shaker at 250 rpm. The flask cultures weretransferred to individual 2 L top drive motor fermenter vessels with aworking volume of 1.2 L of 80 mL of YP medium with 20 g/L glucose, 1%v/v Ethanol, 100 μM CuSO₄.5H₂0, and 0.2 g/L G418 for a starting OD₆₀₀ of0.2. Fermenters were operated at 30° C. and pH 6, controlled with 6N KOHin a two-phase aerobic fermentation. Initially, fermenters were operatedat a growth phase oxygen transfer rate (OTR) of 10 mM/h by fixedagitation of 850 rpm and an air overlay of 5 sL/h. Cultures were grownfor 31 h to approximately 6-7 OD₆₀₀ then immediately switched to aproduction aeration OTR of 0.5 mM/h by reducing agitation from 850 rpmto 300 rpm for the remainder of the fermentation of 111 h. Periodically,samples from each fermenter were removed to measure OD₆₀₀ and to preparefor gas chromatography (GC1) analysis. For GC, 2 mL sample was removedinto an Eppendorf tube and centrifuged in a microcentrifuge for 10 minat maximum. One mL of the supernatant was analyzed by GC1 (isobutanol,other metabolites). At 72 h the same procedures were used to collectcells for OD₆₀₀ and GC analysis and in addition the samples wereanalyzed by high performance liquid chromatography (LC1) for organicacids and glucose.

The results at 111 h are summarized in Table 56. Isobutanol titer,yield, and rate increased with deletion of the TMA29 gene. DH2MBproduction decreased to undetectable levels.

TABLE 56 Isobutanol Titer, Yield, and Rate Increase at 111 h. GlucoseIsobutanol DH2MB Isobutanol consumed^(a) produced^(a) produced^(a)yield^(b) Isobutanol Strain OD₆₀₀ [g/L] [g/L] [g/L] [% theor.] rate^(b)[g/L/h] GEVO3690, 7.2 ± 0.7 29.7 ± 1.1  8.6 ± 0.1 2.9 62.4 ± 3   0.09GEVO3691 (TMA29+) GEVO3694, 7.4 ± 1.3 35.7 ± 3.9 12.3 ± 1.2 0 75.0 ±0.01 0.14 GEVO3695, GEVO3696 (TMA29Δ) ^(a)Glucose, isobutanol, and DH2MBtiters are the final titers, i.e. at 111 h of fermentation.^(b)Isobutanol yield and rate are calculated based on the productionphase only, i.e. from 31 to 111 h of fermentation.

Example 17 Determination of TMA29 Activity in S. cerevisiae

The following example illustrates that the (S)-2-acetolactate reductionactivity is significantly decreased in a tma29Δ strain.

TABLE 57 Genotype of Strains Disclosed in Example 17. GEVO # GenotypeSource GEVO3527 MATα his3Δ-1 leu2Δ ATCC# 201389 (BY4742) lys2Δ ura3ΔGEVO3939 MATα his3Δ-1 leu2Δ OpenBiosystems cat# YSC1054 lys2Δ ura3Δ(Yeast MATalpha tma29::kan^(R) collection)

Yeast strains GEVO3939 from which the TMA29 (YMR226C) gene was deletedand its parent GEVO3527 were each cultured in triplicate by inoculating3 mL of YPD in a 14 mL culture tube in triplicate for each strain.Cultures were started from patches on YPD agar plate for GEVO3527 and onYPD plates containing 0.2 g/L G418 for GEVO3939 and GEVO3940. Thecultures were incubated overnight at 30° C. and 250 rpm. The next day,the OD₆₀₀ of the overnight cultures were measured and the volume of eachculture to inoculate a 50 mL culture to an OD₆₀₀ of 0.1 was calculated.The calculated volume of each culture was used to inoculate 50 mL of YPDin a 250 mL baffled flask and the cultures were incubated at 30° C. and250 rpm.

The cells were harvested during mid-log phase at ODs of 1.6-2.1 after 7h of growth. The cultures were transferred to pre-weighed 50 mL Falcontubes and cells were collected by centrifugation for 5 minutes at3000×g. After removal of the medium, cells were washed with 10 mL MilliQH₂0. After removal of the water, the cells were centrifuged again at3000×g for 5 minutes and the remaining water was carefully removed usinga 1 mL pipette tip. The cell pellets were weighed and then stored at−80° C. until further use.

Cell pellets were thawed on ice and resuspended in lysis buffer (10 mMsodium phosphate pH7.0, 1 mM dithiothreitol, 5% w/v glycerol) such thatthe result was a 20% cell suspension by mass. One mL of glass beads (0.5mm diameter) was added to a 1.5 mL Eppendorf tube for each sample and850 μL of cell suspension were added. Yeast cells were lysed using aRetsch MM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6×1 min eachat full speed with 1 min incubation on ice between. The tubes werecentrifuged for 10 min at 21,500×g at 4° C. and the supernatant wastransferred to a fresh tube. Extracts were held on ice until they wereassayed using the TMA29 assay as described.

The specific activity of S. cerevisiae TMA29 in GEVO3527 lysates, awild-type MATa S. cerevisiae strain, for the reduction of(S)-2-acetolactate was 6.9±0.2 mU/mg. The tma29Δ strain GEVO3939 had aspecific activity of 0.7±0.3 mU/mg. The wild-type GEVO3527 strain hadabout a 10-fold higher specific TMA29 activity than the deletion strain.

Example 18 Determination of TMA29 Activity in Kluyveromyces lactis

The following example illustrates that the (S)-2-acetolactate reductionactivity is significantly decreased in a tma29Δ strain.

TABLE 58 Genotype of Strains Disclosed in Example 18. GEVO # GenotypeGEVO1287 Kluyveromyces lactis, MATα uraA1 trp1 leu2 lysA1 ade1 lac4-8[pKD1] GEVO1742 Kluyveromyces lactis, MATalpha uraA1 trp1 leu2 lysA1ade1 lac4-8 [pKD1] pdc1Δ::kan GEVO4458 Kluyveromyces lactis, MATalphauraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1] pdc1Δ::kan tma29Δ::hph

TABLE 59 Oligonucleotide Sequences Disclosed in Example 18. oGV #Sequence  821 CGGGTAATTAACGACACCCTAGAGG (SEQ ID NO: 132) 2320GGCTGTGTAGAAGTACTCGCCGATAG (SEQ ID NO: 133) 3065AAAAAGGAGTAGAAACATTTTGAAGCTATGCGTTGATAAGGGCAACAACGTTAGTATC (SEQ ID NO: 134) 3066ATACTAACGTTGTTGCCCTTATCAACGCATAGCTTCAAAATGTTTCTACTCCTTTTTTAC (SEQ ID NO: 135) 3067TCAAATTTTTCTTTTTTTTCTGTACAGTTACCCAAGCTGTTTTGCCTATTTTCAAAGC (SEQ ID NO: 136) 3068GCTTTGAAAATAGGCAAAACAGCTTGGGTAACTGTACAGAAAAAAAAGAAAAATTTG (SEQ ID NO: 137) 3069AGTTCAAATCAGTTCGAGGATAATTTAAG (SEQ ID NO: 138) 3070TTAATAAATGCTCAAAAGAAAAAAGGCTGGCG (SEQ ID NO: 139) 3103ACCGGTGCTTCTGCAGGTATTG (SEQ ID NO: 140) 3106ATGCTTGGTTGGAAGCAAATAC (SEQ ID NO: 141)

The K. lactis strain GEVO4458 was constructed from GEVO1742 as follows.DNA constructs were made to delete the TMA29 locus of K. lactis usingSOE PCR. The 5′ targeting sequence was amplified by PCR using GEVO1287genomic DNA as template with primers oGV3103 and oGV3065. The 376 bpfragment was purified by gel electrophoresis. The 3′ targeting sequencewas amplified by PCR using GEVO1287 genomic DNA as template with primersoGV3106 and oGV3067. The 405 bp fragment was gel purified. The Hphmarker was amplified by PCR using pGV2701 (P_(TEF1)-Hph, CEN/ARS,pUC-ori, bla) as template with primers oGV3066 and oGV3068. The 1,165 bpfragment was gel purified. Next the 5′ targeting sequence and the hphmarker were joined together using PCR products described as template.The reaction was amplified using primers oGV3068 and oGV3103. The 1,984bp fragment was gel purified. Next the 5′ targeting sequence plus Hphmarker PCR fragment was joined with the 3′ targeting sequence using PCRwith primers oGV3103 and oGV3106. The 2,331 bp was gel purified and usedfor transformation. Yeast DNA was isolated using the Zymo Research ZRFungal/Bacterial DNA Kit (Zymo Research Orange, Calif.; Catalog #D6005).GEVO1287 was grown to saturation in 12.5 mL of YPD in baffled 125 mLflasks. The entire culture was collected in 15 mL Falcon tubes and cellscollected at 2700 rcf for 5 min. Genomic DNA was isolated according tothe manufacturer's instructions. The DNA concentration was measured andall genomic DNA preps were diluted to a final concentration of 25 ng/μL.

GEVO1742 was transformed as follows. 50 mL YPD medium in 250 mL baffledflasks were inoculated with GEVO1742 cells from a fresh plate. Thecultures were incubated overnight at 30° C. and 250 rpm. The nextmorning the culture was diluted 1:50 in YPD medium and allowed to growfor 6 h. Cells were collected by centrifugation at 2700 rcf for 2 min at30° C. Cells were washed by fully resuspending cells with 50 mL sterileMilliQ water. Cells were collected by centrifugation at 2700 rcf for 2min at 30° C. Cells were washed by resuspending with 25 mL sterileMilliQ water. Cells were collected by centrifugation at 2700 rcf for 2min at 30° C. Cells were resuspended in 1 mL 100 mM lithium acetate,transferred to an Eppendorf tube and collected by centrifuging at 14,000rcf for 10 seconds. The supernatant was removed and the cells wereresuspended with 4× the pellet volume in 100 mM LiOAc. A mixture of DNA(15 μL of PCR product), 72 μL 50% PEG, 10 μL 1 M lithium acetate, and 3μL of denatured salmon sperm DNA (10 mg/mL) was prepared for eachtransformation. In a 1.5 mL tube, 15 μL of the cell suspension was addedto the DNA mixture (170 μL), and the transformation suspension wasvortexed for 5 short pulses. The transformation was incubated for 30 minat 30° C., followed by incubation for 22 min at 42° C. The cells werecollected by centrifugation (18,000×g, 10 sec, 25° C.). The cells wereresuspended in 400 μL YPD medium and allowed to recover overnight at 30°C. and 250 rpm. The following morning, the cells were spread onto YPEplates 1% (w/v) yeast extract, 2% (w/v) peptone, 25 mL/L ethanol)supplemented with 0.1 g/L Hygromycin. Transformants were single colonypurified onto YPE plates supplemented with 0.1 g/L Hygromycin.

The single colony isolates were patched onto YPE supplemented with 0.1g/L Hygromycin plates and the patches were screened for the correctintegration by colony PCR. Presence of the correct PCR product wasconfirmed using agarose gel electrophoresis. To screen for the internalTMA29 coding region, primers oGV3103 and oGV3106 were used. To screenthe 5′ integration junction, primers oGV3069 and oGV821 were used. Toscreen the 3′ integration junction, primers oGV2320 and oGV3070 wereused.

Yeast cells were cultured by inoculating 3 mL of YPD medium (1% (w/v)yeast extract, 2% (w/v) peptone, 2% (w/v) glucose) in a 14 mL culturetube in triplicate for each strain. Cultures were started from patcheson a YPD plate 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v)glucose, 2% agar). The cultures were incubated overnight at 30° C. and250 rpm. The next day, the OD₆₀₀ of the overnight cultures were measuredand the volume of each culture to inoculate a 50 mL culture to an OD₆₀₀of 0.1 was calculated. The calculated volume of each culture was used toinoculate 50 mL of YPD in a 250 mL baffled flask and the cultures wereincubated at 30° C. and 250 rpm overnight. Cells were harvested duringmid-log phase at ODs of 1.8-2.2. The cultures were transferred topre-weighed 50 mL Falcon tubes and cells were collected bycentrifugation for 5 min at 3000×g. After removal of the medium, cellswere washed with 10 mL MilliQ H₂0. After removal of the water, the cellswere centrifuged again at 3000×g for 5 min and the remaining water wascarefully removed with a 1 mL pipette tip. The cell pellets were weighedand then stored at −80′C.

Cell pellets were thawed on ice and resuspended in lysis buffer (10 mMsodium phosphate pH7.0, 1 mM dithiothreitol, 5% w/v glycerol) such thatthe result was a 20% cell suspension by mass. One mL of glass beads (0.5mm diameter) was added to a 1.5 mL Eppendorf tube for each sample and850 μL of cell suspension were added. Yeast cells were lysed using aRetsch MM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6×1 min eachat full speed with 1 min incubation on ice between. The tubes werecentrifuged for 10 min at 21,500×g at 4° C. and the supernatant wastransferred to a fresh tube. Extracts were held on ice until they wereassayed using the TMA29 assay as described.

The specific activity of Gevo1742 with the TMA29 gene for the reductionof (S)-2-acetolactate was 0.0043±0.0005 μmol/min/mg lysate. The specificactivity of Gevo4459 deleted for the TMA29 gene was 0.0019±0.0003μmol/min/mg lysate.

Example 19 Increased Isobutanol Yield in Strains Comprising an ALD6Deletion, a TMA29 Deletion and an Alcohol Dehydrogenase with Increasedk_(Cat) and Decreased K_(m) in S. cerevisiae

The following example illustrates that the combination of an ALD6deletion, TMA29 deletion and overexpression of a gene encoding an ADHwith improved kinetic properties leads to increased isobutanolproduction and theoretical yield.

A S. cerevisiae CEN.PK2 strain, GEVO3991, was constructed bytransforming a S. cerevisiae CEN.PK2 strain, GEVO3956, which expressesan improved alcohol dehydrogenase (L. lactis ADH*, LI_ADH*) and adecarboxylase (L. lactis KIVD, LI_kivD2) from its chromosomal DNA with a2μ plasmid, pGV2603 (P_(TDH3):Ec_ilvC_coSc^(P2D1-A1-his6),P_(TEF1):LI_ilvD_coSc, P_(ENO2):LI_adhA^(RE1), 2μ-ori, pUC-ori, bla,G418R), expressing genes encoding enzymes: KARI, DHAD, and the improvedADH (EC_ilvC_coSc^(P2D1-A1-his6), LI_ilvD_coSc, and LI_adhA^(RE1),respectively).

TABLE 60 Genotype of Strains Disclosed in Example 19. GEVO No. GenotypeGEVO3991 MATa ura3 leu2 his3 trp1 ald6Δ::[P_(ENO2): Ll_adhA^(RE1):P_(FBA1): Sc_ TRP1 gpd1Δ::T_(KI) _(—) _(URA3) gpd2Δ::T_(KI) _(—) _(URA3)tma29Δ::T_(KI) _(—) _(URA3) pdc1Δ::[P_(PDC1): Ll_kivD2_coSc5: P_(FBA1):LEU2: T_(LEU2): P_(ADH1): Bs_alsS1_coSc: T_(CYC1): P_(PGK1):Ll_kivD2_coEc: P_(ENO2): Sp_HIS5] pdc5Δ::T_(KI) _(—) _(URA3)pdc6Δ::P_(TDH3): Sc_AFT1: P_(ENO2): Ll_adhA^(RE1): T-_(KI) _(—) _(URA3)_(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)]{evolved for C2supplement-independence, glucose tolerance and faster growth}, [pGV2603]GEVO3956 MATa ura3 leu2 his3 trp1 ald6Δ::[P_(ENO2): Ll_adhA^(RE1):P_(FBA1): Sc_TRP1 gpd1Δ::T_(KI) _(—) _(URA3) gpd2Δ::T_(KI) _(—) _(URA3)tma29Δ::T_(KI) _(—) _(URA3) pdc1Δ::[P_(PDC1): Ll_kivD2_coSc5: P_(FBA1):LEU2: T_(LEU2): P_(ADH1): Bs_alsS1_coSc: T_(CYC1): P_(PGK1):Ll_kivD2_coEc: P_(ENO2): Sp_HIS5] pdc5Δ::T_(KI) _(—) _(URA3)pdc6Δ::P_(TDH3): Sc_AFT1: P_(ENO2): Ll_adhA^(RE1): T-_(KI) _(—) _(URA3)_(—) _(short): P_(FBA1): KI_URA3: T_(KI) _(—) _(URA3)]{evolved for C2supplement-independence, glucose tolerance and faster growth}

A fermentation was performed to determine the performance of GEVO3991(LI_adhA^(RE1), ALD6Δ, TMA29Δ) in four replicate fermenters. Glucoseconsumption, isobutanol production, isobutyrate production, acetateproduction and OD₆₀₀ were measured during the fermentation. For thesefermentations, single isolate cell colonies grown on YPD agar plateswere transferred to 500 mL baffled flasks containing 80 mL of YPDcontaining 80 g/L glucose, 5 g/L ethanol, 0.5 g/L MgSO₄, and 0.2 g/LG418 and incubated for 30 h at 30° C. in an orbital shaker at 250 rpm.The flask cultures were transferred to four individual 2 L top drivemotor fermenter vessels with a working volume of 0.9 L of YPD containing80 g/L glucose, 5 g/L ethanol, 0.5 g/L MgSO₄, and 0.2 g/L G418 pervessel for a starting OD₆₀₀ of 0.3. Fermenters were operated at 30° C.and pH 6.0 controlled with 6N KOH in a 2-phase aerobic condition basedon oxygen transfer rate (OTR). Initially, fermenters were operated at agrowth phase OTR of 10 mM/h by fixed agitation of 700 rpm and an airoverlay of 5sL/h. Cultures were grown for 22.5 h to approximately 10-11OD₆₀₀ then immediately switched to production aeration conditions for40.7 h. Cell density during production phase approached 13-14 OD₆₀₀. Theproduction phase was operated at an OTR of 0.5 mM/h by fixed agitationof 300 rpm. Periodically, samples from each fermenter were removed tomeasure OD₆₀₀ and to prepare for gas chromatography (GC) and liquidchromatography (LC) analysis. For GC and LC, 2 mL sample was removedinto an Eppendorf tube and centrifuged in a microcentrifuge for 10 minat maximum. One mL of the supernatant was analyzed by GC1 (isobutanol,other metabolites) and one mL analyzed by high performance liquidchromatography (LC1) for organic acids and glucose as described.

GEVO3991 achieved a cell density of 13.8 during the 22.5 h growth phase.The isobutanol produced during the entire duration of the experiment(63.2 h) was 18.6±0.9 g/L with 0.84±0.10 g/L isobutyrate and 0.15±0.02g/L acetate produced. The theoretical isobutanol yield achieved duringthe production phase of the experiment (22.5-63.5 h) was 80.3±1.1% whilethe isobutyrate yield was only 0.013±0.001 g/g glucose. The productionof DH2MB was not detected.

In addition, three independent transformants of GEVO3991 were alsocharacterized in shake flasks. The strain was grown overnight in 3 mL ofYPD containing 1% ethanol and 0.2 g/L G418 at 30° C. at 250 rpm. Thesecultures were diluted to an OD₆₀₀ of 0.1 in 50 mL of the same medium ina baffled 250 mL flask and grown overnight. The OD₆₀₀ was measured and avolume of cells approximately equal to 250 OD₆₀₀ was collected for eachculture by centrifugation at 2700 rcf for 2 minutes and the cells wereresuspended in 50 mL of fermentation medium (YPD containing 80 g/Lglucose, 0.03 g/L ergosterol, 1.32 g/L Tween80, 1% v/v ethanol, 200 mMMES, pH6.5), and transferred to an unbaffled vented screw cap 250 mLflask. The OD₆₀₀ was checked and the cultures were placed at 30° C. at75 rpm to initiate the microaerobic fermentation. Samples for liquidchromatography (LC), gas chromatography (GC) analysis and OD₆₀₀ weretaken at roughly 24 h intervals. The samples (2 mL) were centrifuged at18,000×g for 10 min and 1.5 mL of the clarified supernatant was used foranalysis by GC1 and LC1.

Fermentations started at an OD₆₀₀ of about 4. The cells grew to an OD₆₀₀of about 8 by 72 h of microaerobic fermentation. After 72 h, theisobutanol titer was 12.3 g/L and the isobutanol yield was 67.2% oftheoretical. Isobutyrate titer and yield were low: 0.6 g/L isobutyratewas produced at a yield of 0.013 g/g glucose. The production of DH2MBwas not detected.

Example 20 Effect of TMA29 Deletion in K. marxianus

The purpose of this example is to demonstrate that the deletion of TMA29in a Kluyveromyces marxianus strain comprising ALS activity results inreduced DH2MB production.

Strains, plasmids, and oligonucleotide sequences disclosed in thisexample are listed in Tables 61, 62, and 63, respectively.

TABLE 61 Genotype of Strains Disclosed in Example 20. GEVO No. Genotype1947 ura3-delta2, derived from strain NRRL-Y-7571 Kluyveromycesmarxianus (E. C. Hansen) van der Walt (1971) 2348 ura3-delta2pdc1Δ::G418R, P_(Sc) _(—) _(PDC1): 31COX4 MTS: Bs_alsS: P_(Sc) _(—)_(FBA1): URA3 ura3-delta2 6403, 6404 ura3-delta2 pdc1Δ::G418R, P_(Sc)_(—) _(PDC1): 31COX4 MTS: alsS: P_(Sc) _(—) _(FBA1): URA3 ura3-delta2tma29Δ::P_(Sc) _(—) _(TEF1)-hph

TABLE 62 Plasmid Disclosed in Example 20. Plasmid Name RelevantGenes/Usage Genotype pGV2701 For SOE PCR to give P_(TEF1): hph, CEN, thehph fragment pUC ori, bla

TABLE 63 Oligonucleotide Sequences Disclosed in Example 20. PrimerSequence 3498 ATGTCTCAAGGTAGAAGAGCTG (SEQ ID NO: 142) 3137GGAGTAGAAACATTTTGAAGCTATGTATATCTTCTGAATCAATTGCACCGAC (SEQ ID NO: 143)3140 CAAATTTTTCTTTTTTTTCTGTACAGAGAGGTATGATTAATACCAATGTCTTGGG(SEQ ID NO: 144) 3499 TCATTCACCACGGTAAATGTGG (SEQ ID NO: 145) 3138GTCGGTGCAATTGATTCAGAAGATATACATAGCTTCAAAATGTTTCTACTCC (SEQ ID NO: 146)3139 GTATTAATCATACCTCTCTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACG(SEQ ID NO: 147) 3501GAAGGAAATTCCAGTCTCCTAGTTCCTTTGAACAC (SEQ ID NO: 148) 2320GGCTGTGTAGAAGTACTCGCCGATAG (SEQ ID NO: 149) 3500CAGAACAATCAATCAACGAACGAACGACCCACCC (SEQ ID NO: 150)  821CGGGTAATTAACGACACCCTAGAGG (SEQ ID NO: 151) 3141AAGGAGATGCTTGGTTTGTAGCAAACACC (SEQ ID NO: 152)

Strain Construction: The K. marxianus TMA29 gene homolog encoding the K.marxianus TMA29 protein (SEQ ID NO: 23) was deleted from parent K.marxianus strain GEVO2348 as follows, resulting in strains GEVO6403 andGEVO6404.

Genomic DNA was isolated from GEVO1947 as described. Constructs weremade to integrate the E. coli hph (hygromycin resistance) cassette intothe TMA29 locus of GEVO2348 by SOE PCR as described. PCR step #1consisted of three reactions resulting in the 5′ TMA29 targetingsequence, the 3′ TMA29 targeting sequence, and the hph marker. The 5′targeting sequence was amplified from prepared GEVO1947 genomic DNA withprimers oGV3498 and oGV3137. The 385 bp fragment was purified by gelelectrophoresis. The 3′ targeting sequence was amplified from preparedGEVO1947 genomic DNA with primers oGV3140 and oGV3499. The 473 bpfragment was gel purified. The P_(TEF1):hph:T_(CYC1(partial)) cassettewas amplified from pGV2701 with primers oGV3138 and oGV3139. The 1,651bp fragment was gel purified. The final SOE PCR step joined the 3products from step #1 (5′ targeting sequence/hph marker/3′ targetingsequence). The reaction was amplified using primers oGV3498 and oGV3499.The 2,414 bp fragment was gel purified as described and used fortransformation of GEVO2348 as described. Medium used to grow the cellsfor the transformation was YPE. Following the transformation, 150 μL ofthe transformation culture was spread onto YPE plates containing 0.1 g/Lhygromycin. The plates were incubated at 30° C. and transformed colonieswere single colony isolated and then patched for colony PCR on YPEplates containing 0.1 g/L hygromycin.

Yeast Colony PCR was used to screen for the appropriate 3′ integrationjunction, 5′ integration junction, as well as lack of the TMA29 codingregion as described. The proper 3′ integration junction was confirmedusing primers oGV3501 and 2320. The proper 5′ integration junction wasconfirmed using primers oGV3500 and oGV0821 were used. Finally, toscreen for deletion of the TMA29 internal coding region, primers oGV3500and oGV3141 were used.

Fermentation: Shake flask fermentations was performed in triplicate foreach of the strains GEVO2348 (TMA29), GEVO6403 (tma29Δ), and GEVO6404(tma29Δ) as described to determine if deletion of TMA29 in strainsexpressing Bs_alsS would result in diminished production of DH2MB.Single colony isolated transformants of tma29Δ strains were patched toYPE plates containing 0.1 g/L hygromycin, while parent strains werepatched to YPE plates. Cells from the patches were used to inoculate 3mL cultures of YPE. Cultures were incubated overnight at 30° C. and 250rpm. After overnight incubation, the OD₆₀₀ of these cultures wasdetermined by diluting 1:40 in water. The appropriate amount of culturewas added to 50 mL of YPE to obtain an OD₆₀₀ of 0.1 in 250 mL baffledflasks and incubated at 30° C. and 250 rpm. After a 24 h incubation, theOD₆₀₀ of these cultures was determined by diluting 1:40 in water. Theappropriate amount of culture was added to 50 mL of YPD containing 8%glucose and 200 mM MES, pH 6.5 to obtain an OD₆₀₀ of 5. Fermentationcultures were incubated at 30° C. and 75 rpm in unbaffled 250 mL flasks.One 15 mL aliquot of medium was also collected to use as a blank for LC4analysis and was kept at 4° C. until sample submission. After 72 h, 1.5mL of culture was removed and samples were prepared as above for OD₆₀₀and LC4 analysis. In addition, samples for enzyme assays were harvestedat 72 h by transferring 80 OD's of the appropriate sample to two 15 mLFalcon tubes centrifuged at 3000×g for 5 min at 4° C. Pellets wereresuspended in 3 mL cold, sterile water and were centrifuged at 5000×gfor 2 min at 4° C. in a swinging bucket rotor in the tabletopcentrifuge. The water was removed by vacuum aspirator. The conical tubeswere stored at −80° C.

The in vitro ALS enzymatic activities of the lysates were measured asdescribed. Table 64 shows the average in vitro ALS enzymatic activity oflysates from the strains after 72 h. ALS activity is measurable inGEVO2348 (average of 3.14 Units/mg lysate) as well as in both tma29Δstrains GEVO6403 and GEVO6404 (averages of 1.63 and 1.58 Units/mg lysaterespectively).

Table 64 also shows the DH2MB and DHIV titers by LC4 for these strains.GEVO2348 (TMA29) strains produced average DH2MB titers of 0.89 g/L whileDHIV was not detected. The DH2MB titers were significantly decreased inthe tma29Δ strains GEVO6403 and GEVO6404 which measured at 0.16 and 0.15g/L respectively. While the ALS activity is decreased in the tma29Δstrains, this does not account for the >80% decrease in DH2MB titers inthe deletion strains. For example, one technical replicate of GEVO2348exhibited an ALS activity of 2.5 Units/mg lysate and produced 0.83 g/LDH2MB while one of the technical replicates of the tma29Δ strainGEVO6404 has similar activity of 1.9 Units/mg lysate and produced only0.16 g/L DH2MB.

TABLE 64 ALS Activity, DH2MB and DHIV titers, and Percent DH2MB Decreasein tma29Δ Strains After 72 h Fermentation. ALS DH2MB DHIV DH2MB Activity(U/ by LC4 by LC4 decrease Strain TMA29 mg lysate) (g/L) (g/L) (%)GEVO2348 + 3.1 ± 0.5 0.89 ± 0.07 n.d. GEVO6403 Δ 1.6 ± 0.2 0.16 ± 0.02n.d. 82% GEVO6404 Δ 1.6 ± 0.3 0.15 ± 0.01 n.d. 83% n.d. = not detected

Example 21 Effect of TMA29 Deletion in Kluyveromyces lactis

The purpose of this example is to demonstrate that the deletion of TMA29in a Kluyveromyces lactis strain comprising ALS activity results inreduced DH2MB production.

Strains, plasmids, and oligonucleotide primers disclosed in this exampleare listed in Tables 65, 66, and 67, respectively.

TABLE 65 Genotype of Strains Disclosed in Example 21. GEVO NumberGenotype 1742 MATalpha uraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1]pdc1::kan, derived from K. lactis strain ATCC 200826 (Kluyveromyceslactis (Dombrowski) van der Walt, teleomorph) 4458 MATalpha uraA1 trp1leu2 lysA1 ade1 lac4-8 [pKD1] pdc1::kan tma29::hph 6310, 6311, MATalphauraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1] 6312 pdc1::kan [pGV1429] 6313,6314, MATalpha uraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1] 6315 pdc1::kan[pGV1645] 6316, 6317 MATalpha uraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1]pdc1::kan + random integration of Bs_alsS: TRP1 6318, 6319, MATalphauraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1] 6320 pdc1::kan tma29::hph[pGV1429] 6321, 6322, MATalpha uraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1]6323 pdc1::kan tma29::hph [pGV1645] 6324, 6325 MATalpha uraA1 trp1 leu2lysA1 ade1 lac4-8 [pKD1] pdc1::kan tma29::hph + random integration ofBs_alsS: TRP1

TABLE 66 Plasmids Disclosed in Example 21. Plasmid Name RelevantGenes/Usage Genotype pGV1429 High copy 1.6μ empty 1.6 μ-ori, PMB1 ori,vector containing TRP1 bla, TRP1 pGV1645 High copy 1.6μ vector 1.6μ-ori, PMB1 ori, containing TRP1 and bla, TRP1, Bs_alsS Bs_alsS pGV1726Vector containing TRP1 PMB1 ori, bla, TRP1, (linearized and Bs_alsSBs_alsS with AhdI)

TABLE 67 Oligonucleotide Sequences Disclosed in Example 21. PrimerSequence oGV3065 AAAAAGGAGTAGAAACATTTTGAAGCTATGCGTTGATAAGGGCAACAACGTTAGTATC (SEQ ID NO: 153) oGV3066ATACTAACGTTGTTGCCCTTATCAACGCATAGCTTCAAAATGTTTCTACTCCTTTTTTAC (SEQ ID NO: 154) oGV3067TCAAATTTTTCTTTTTTTTCTGTACAGTTACCCAAGCTGTTTTGCCTATTTTCAAAGC (SEQ ID NO: 155) oGV3068GCTTTGAAAATAGGCAAAACAGCTTGGGTAACTGTACAGAAAAAAAAGAAAAATTTG (SEQ ID NO: 156) oGV3103 ACCGGTGCTTCTGCAGGTATTG (SEQ ID NO: 157)oGV3106 ATGCTTGGTTGGAAGCAAATAC (SEQ ID NO: 158) oGV1321AATCATATCGAACACGATGC (SEQ ID NO: 159) oGV1324AGCTGGTCTGGTGATTCTAC (SEQ ID NO: 160)

Strain Construction: The K. lactis TMA29 gene homolog encoding the K.lactis TMA29 protein (SEQ ID NO: 7) was deleted from parent K. lactisstrain GEVO1742 as follows, resulting in strain GEVO4458 as described inExample 18.

K. lactis strains GEVO1742 (parent, TMA29) and GEVO4458 (tma29Δ) weretransformed with plasmid pGV1429 (empty control vector), pGV1645(expressing Bs_alsS) or with AhdI linearized plasmid pGV1726 (resultingin random integration of Bs_alsS) as described, resuspended in 400 μL of1.25×SC-HWLU and spread over SCD-W plates to select for transformedcells. Random integration of AhdI linearized pGV1726 in both GEVO1742and tma29Δ strain GEVO4458 was confirmed by colony PCR with primersoGV1321 and oGV1324 that are specific to the internal Bs_alsS codingregion as described. Strains GEVO6316, GEVO6317, GEVO6324, and GEVO6325were positive for the gene integration.

Fermentation: A shake flask fermentation was performed on the variousGEVO strains (Table 65) as described to determine if deletion of TMA29in strains expressing Bs_alsS would result in diminished production ofDH2MB. Single colony isolated transformants were patched to SCD-Wplates, non transformed parents were patched onto YPD. Cells from thepatches were used to inoculate 3 mL cultures in either YPD (parentstrains and integrated strains) or 3 mL SCD-W. Cultures were incubatedovernight at 30° C. and 250 rpm. After overnight incubation, the OD₆₀₀of these cultures was determined by diluting 1:40 in water. Theappropriate amount of culture was added to 50 mL of YPD containing 5%glucose or SCD-W containing 5% glucose to obtain an OD₆₀₀ of 0.1 in 250mL baffled flasks and incubated at 30° C. and 250 rpm. After 24 hincubation, the OD₆₀₀ of these cultures was determined by diluting 1:40in water. The appropriate amount of culture was added to 50 mL of YPDcontaining 8% glucose, 200 mM MES pH 6.5 or SCD-W containing 8% glucoseto obtain an OD₆₀₀ of 5. When 250 OD's were not available to start thefermentation, the entire 50 mL culture was used. Fermentation cultureswere incubated at 30° C. and 75 rpm in unbaffled 250 mL flasks. A 15 mLconical tube was also collected for media blanks for LC1 and LC4analysis as described and kept at 4° C. until sample submission. At the72 h timepoint, 1.5 mL of culture was collected. OD₆₀₀ values weredetermined and samples were prepared for LC1 and LC4 analysis bycentrifuging for 10 min at 14,000 rpm and removing 1 mL of thesupernatant to be analyzed. In addition samples for enzyme assays wereharvested at the 72 h timepoint. 60 OD's of the appropriate sample weretransferred into a 15 mL Falcon tube and centrifuged at 3000×g for 5 minat 4° C. Pellets were resuspended in 3 mL cold, sterile water andtransferred to 3, 1.5 mL Eppendorf tubes (1 mL each) to make 3×20 ODreplicates. The tubes were centrifuged at 5000×g for 2 min at 4° C. in aswinging bucket rotor in the tabletop centrifuge. The water was removedby vacuum aspirator. The Eppendorf tubes were stored at −80° C.

The in vitro ALS enzymatic activities of the lysates were measured asdescribed. Table 68 shows the average in vitro ALS enzymatic activity oflysates from the strains after 72 h. ALS activity was measurable only instrains with Bs_alsS randomly integrated (GEVO6316, GEVO6317, GEVO6324,6325) or expressed from plasmid (GEVO6313-6315, GEVO6321-6323). ALSactivity in strains with Bs_alsS integrated is lower than in strainsexpressing Bs_alsS from plasmid. However, the activity of 0.25 Units/mglysate in the TMA29 strains with integrated Bs_alsS (GEVO6316, GEVO6317)was still enough to produce a titer 1.06 g/L of combined DHIV+DH2MB.

Table 68 shows the combined DHIV+DH2MB titers for the various strainsafter 72 h of fermentation based on LC1 analysis. Strain GEVO1742(parent, TMA29) strains produced measurable combined DHIV+DH2MB titersonly when Bs_alsS was randomly integrated (1.06 g/L) or expressed fromplasmid pGV1645 (0.45 g/L). These DHIV+DH2MB titers were abolished inthe tma29Δ strain GEVO4458 when expressing Bs_alsS via randomintegration (GEVO6324, GEVO6325) or plasmid (GEVO6321-6323). LC4analysis indicated that the majority of the combined DHIV+DH2MB titerwas in fact DH2MB.

TABLE 68 ALS Activity, Combined DHIV + DH2MB Titer, and Percentage ofDH2MB of Combined DHIV + DH2MB Titer. Plasmid Integrated (I), DHIV +DH2MB % DH2MB in Parent plasmid (P), ALS Activity by LC1 DH2MB + DHIVStrain Strain or control (C) TMA29 ALS (U/mg lysate) (g/L) by LC4GEVO1742 none + − 0.00 ± 0.00 0.00 ± 0.00 n/a GEVO6316, 6317 GEVO1742pGV1726 (I) + + 0.25 ± 0.06 1.06 ± 0.23 80.0 ± 3.7 GEVO4458 GEVO1742none Δ − 0.00 ± 0.00 0.00 ± 0.00 n/a GEVO6324, 6325 GEVO4458 pGV1726 (I)Δ + 0.86 ± 0.28 0.00 ± 0.00 n/a GEVO6310-6312 GEVO1742 pGV1429 (C) + −0.00 ± 0.00 0.00 ± 0.00 n/a GEVO6313-6315 GEVO1742 pGV1645 (P) + + 6.12± 1.09 0.45 ± 0.02 87.2 ± 2.3 GEVO6318-6320 GEVO4458 pGV1429 (C) Δ −0.00 ± 0.00 0.00 ± 0.00 n/a GEVO6321-6323 GEVO4458 pGV1645 (P) Δ + 1.23± 0.45 0.00 ± 0.00 n/a n/a = not applicable, samples had no detectablepeak by LC1 so were not analyzed by LC4

Example 22 Effect of TMA29 Deletion in I. orientalis

The following example illustrates that deletion of the I. orientalisTMA29 gene results in decreased TMA29 activity and also results indecrease in DH2MB production in strains comprising ALS activity.

TABLE 69 Genotype of Strains Disclosed in Example 22. GEVO # RelevantGenotype GEVO4450 ura3Δ/ura3Δ pdc1-1Δ::Ll_kivD: T_(ScCYC1): loxP:P_(ENO1): Bs_alsS/ pdc1-2Δ::Ll_kivD: T_(ScCYC1): loxP: P_(ENO1): Bs_alsSTMA29/TMA29 GEVO12425 ura3Δ/ura3Δ pdc1-1Δ:: Ll_kivD: T_(ScCYC1): loxP:P_(ENO1): Bs_alsS pdc1-2Δ:: Ll_kivD: loxP TMA29/TMA29 GEVO6155ura3Δ/ura3Δ pdc1-1Δ::Ll_kivD: T_(ScCYC1): loxP: P_(ENO1): Bs_alsSpdc1-2Δ::Ll_kivD: loxP TMA29/ tma29Δ::P_(PDC): Ll_adhA^(RE1): P_(TDH3):Ec_ilvC^(P2D1-A1): loxP: URA3: loxP: P_(ENO1): Ll_ilvD GEV06158ura3Δ/ura3Δ pdc1-1Δ:: Ll_kivD: T_(ScCYC1): loxP: P_(ENO1): Bs_alsSpdc1-2Δ:: Ll_kivD: loxP tma29Δ:: P_(PDC): Ll_adhA^(RE1): P_(TDH3):Ec_ilvC^(P2D1-A1)_coCB: loxP: URA3: loxP: P_(ENO1): Ll_ilvD/ tma29Δ::P_(PDC): Ll_adhA^(RE1): P_(TDH3): Ec_ilvC^(P2D1-A1): loxP: URA3: loxP:P_(ENO1): Ll_ilvD GEVO12473 ura3Δ/ura3Δ pdc1-1Δ:: Ll_kivD: T_(ScCYC1):loxP: P_(ENO1): Bs_alsS pdc1-2Δ:: Ll_kivD: loxP tma29Δ:: loxP: URA3:loxP/ tma29Δ::loxP: MEL5: loxP GEVO12474 ura3Δ/ura3Δ pdc1-1Δ:: Ll_kivD:T_(ScCYC1): loxP: P_(ENO1): Bs_alsS pdc1-2Δ:: Ll_kivD: loxP tma29Δ::loxP: URA3: loxP/ tma29Δ:: loxP: MEL5: loxP

Strain Construction: Issatchenkia orientalis strains derived fromPTA-6658 were constructed that were wild-type for the TMA29 gene(GEVO4450, GEVO12425), heterozygous for deletion of one copy of theTMA29 gene (GEVO6155), or completely deleted for the TMA29 gene(GEVO6158, GEVO12473, GEVO12474) using standard yeast genetics andmolecular biology methods. These strains also carry a copy of theBacillus subtilis alsS gene.

TMA29 Enzyme Assay: For the TMA29 in vitro assay, I. orientalis strainsGEVO4450 (TMA29/TMA29), GEVO6155 (tma29Δ/TMA29), and GEVO6158 (completetma29Δ/tma29Δ) were grown by inoculating 25 mL YPD in 125 mL baffledflasks with cells from a fresh YPD plate. Cultures were grown overnightat 30° C. and 250 rpm. These cultures were used to inoculate 50 mL ofYPD in 250 mL baffled flasks to an OD₆₀₀ of 0.05. The cultures weregrown at 30° C. and 250 rpm until they had reached an OD₆₀₀ ofapproximately 5-8 (late log phase). Cells were harvested by collecting80 ODs of cells in a 50 mL Falcon tube and centrifuging at 2,700×g for 3min. After removal of supernatant, cells were placed on ice and washedwith 5 mL cold water. Cells were centrifuged at 2,700×g for 3 min andthe water was removed. The cell pellets were stored at −80° C. untiluse. Additionally, the same strains were grown by inoculating 3 mL ofYPD from fresh plates and growing for 8 h at 30° C. and 250 rpm. Thesecultures were used to inoculate 50 mL of YPD in 250 mL baffled flasks toan OD₆₀₀ of 0.01 and the cultures were grown at 30° C. and 250 rpm untilthey reached an OD₆₀₀ of approximately 4-8. This culture was used toinoculate 50 mL of YPD containing 8% glucose, 200 mM MES pH 6.5 to afinal OD₆₀₀ of 4-5 by centrifuging an appropriate amount of culture at2,700×g for 3 min in a 50 mL Falcon tube and then resuspending the cellpellet in 50 mL of the stated medium. Cells were incubated in 250 mLnon-baffled flasks at 30° C. and 75 rpm for 48 h (fermentation phase).Eighty OD cell pellets were harvested as described. Cells wereresuspended, lysed and assayed for TMA29 activity as described.

Table 70 shows the specific TMA29 activity of lysates of I. orientalisstrains GEVO4450, 6155, and 6158 in U/mg of total protein. SpecificTMA29 activity is reduced in GEVO6155 (tma29/TMA29) and GEVO6158(complete tma29 deletion) as compared to GEVO4450 (TMA29/TMA29).

TABLE 70 TMA29 Activity in I. orientalis Strains. TMA29 activity TMA29activity Late log phase 48 h fermentation phase STRAIN [U/mg totalprotein] [U/mg total protein] GEVO4450 0.0048 ± .0010 .0027 ± .0003GEVO6155 0.0025 ± .0008 .0010 ± .0001 GEVO6158 0.0023 ± .0003 .0010 ±.0003

Fermentation: For the fermentation, I. orientalis strains GEVO12425(TMA29/TMA29), GEVO12473 (tma29/tma29), and GEVO12474 (tma29/tma29) weregrown by inoculating 12 mL YPD in 125 mL baffled flasks with cells froma fresh YPD plate. Cultures were grown overnight at 30° C. and 250 rpm.The OD₆₀₀ of the 12 mL overnight cultures were determined and theappropriate amount was used to inoculate 50 mL YPD containing 5% glucosein 250 mL baffled flasks to an OD₆₀₀ of 0.1. The flasks were incubatedat 30° C. and 250 rpm overnight. The OD₆₀₀ of the 50 mL cultures wasdetermined. The appropriate amount of culture was centrifuged at 2700rcf for 5 min at 25° C. in 50 mL Falcon tubes and the supernatantremoved. The cells from each 50 mL culture were resuspended in 50 mL YPDcontaining 8% glucose, 200 mM MES, pH 6.5. The cultures were thentransferred to 250 mL unbaffled screw-cap flasks and incubated at 30° C.and 75 rpm. At 72 h samples from each flask were removed, the OD₆₀₀ wasmeasured and samples prepared for LC4 analysis by transferring 1 mLsample to an Eppendorf tube and centrifuging at 18,000×g, 10 seconds,25° C. After centrifugation, 0.75 mL of supernatant was transferred to amicrotiter plate and analyzed by LC4. Also at 72 h cells for enzymeassays were collected by transferring 80 ODs to 15 mL Falcon tubes asdescribed. Cells for ALS assays were resuspended, lysed, and assayed asdescribed.

Table 71 shows the DH2MB production and ALS activities for GEVO12425,12473, and 12474 at 72 h. The DH2MB titer was determined by LC4. The ALSactivity was similar in all strains.

TABLE 71 DH2MB Production and ALS Activity in I. orientalis Strains at72 h Fermentation. DH2MB by ALS activity STRAIN LC4 [g/L] [U/mg]GEVO12425 1.87 ± 0.60 4.6 ± 1.1 GEVO12473 0.08 ± 0.01 4.0 ± 0.1GEVO12474 0.07 ± 0.00 3.1 ± 1.1

Example 23 Effect of TMA29 Deletion in S. pombe

The following example illustrates that the (S)-2-acetolactate reductionactivity is significantly decreased in an S. pombe tma29Δ straincompared to an S. pombe TMA29 strain.

TABLE 72 Genotype of strains disclosed in Example 23. GEVO # GenotypeSource GEVO6444 h⁺ ade6-M216, ura4-D18, leu1-32 Bioneer strain BG_0000H8GEVO6445 h+ SPAC521.03Δ::kanMX4, Bioneer strain ade6-M216, ura4-D18,leu1-32 BG_1772H TMA29 homolog (SEQ ID NO: 22) deleted

Yeast strains GEVO6444 which has an intact TMA29 gene (SEQ ID NO: 161)and GEVO6445 which has the TMA29 gene deleted, were grown overnight in12 mL YPD in 125 mL baffled flasks at 250 rpm and 30° C. The next day,OD₆₀₀ values were determined and technical triplicate cultures werestarted in 50 mL YPD with 5% glucose at an OD₆₀₀ of approximately 0.3.Cultures were allowed to grow at 250 rpm and 30° C. throughout the day.At the end of the day, the cultures were diluted in YPD with 5% glucoseto an OD₆₀₀ of approximately 0.15 and incubated overnight at 250 rpm and30° C. The cells were harvested upon reaching an OD₆₀₀ of between 4 and6. To harvest pellets for enzyme assays 80 ODs of the appropriate samplewere transferred into two 15 mL Falcon tube (for duplicate samples) andcentrifuged at 3000×g for 5 min at 4° C. Pellets were resuspended in 3mL cold, sterile water and were centrifuged at 5000×g for 2 min at 4° C.in a swinging bucket rotor in the tabletop centrifuge. The water wasremoved by vacuum aspirator. The pellets were stored at −80° C. Lysateswere prepared and TMA29 enzyme assays were performed as described.

The specific activity of S. pombe GEVO6444 lysates for the reduction of(S)-2-acetolactate was 0.018±0.002 U/mg total protein. Lysates of thetma29Δ strain GEVO6445 had a specific activity of 0.001±0.002 U/mg totalprotein.

Example 24 Effect of ALD6 Deletion in K. marxianus

The purpose of this example is to demonstrate that the deletion of ALD6in a Kluyveromyces marxianus strain results in reduced isobutyraldehydeoxidation activity and isobutyrate production.

Strains, plasmids, and oligonucleotide primers disclosed in this exampleare listed in Tables 73, 74, and 75, respectively

TABLE 73 Genotype of K. marxianus Strains Disclosed in Example 24. GEVONumber Genotype GEVO1947 ura3-delta2 GEVO6264, ura3-delta2ald6Δ::P_(TEF1)-hph GEVO6265 GEVO2087 ura3-delta2, PDC1, P_(Sc) _(—)_(PDC1): 31COX4 MTS: alsS: P_(Sc) _(—) _(TDH3): kivD co HMI1 MTS: P_(Sc)_(—) _(ADH1): ADH7: P_(Sc) _(—) _(FBA1): URA3 GEVO6270 ura3-delta2,PDC1, P_(Sc) _(—) _(PDC1): 31COX4 MTS: alsS: GEVO6271 P_(Sc) _(—)_(TDH3): kivD co HMI1 MTS: P_(Sc) _(—) _(ADH1): ADH7: P_(Sc) _(—)_(FBA1): URA3 ald6Δ::P_(TEF1)-hph

TABLE 74 Plasmids Disclosed in Example 24. Plasmid Name RelevantGenes/Usage Genotype pGV2701 For SOE PCR to P_(TEF1): hph, CEN, give thehph fragment pUC ori, bla

TABLE 75 Oligonucleotide Sequences Disclosed in Example 24. PrimerSequence oGV3490 GTCAAGATTGTTGAACAAAAGCC (SEQ ID NO: 162) oGV3492GAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGGTTTAGTGGGGTTGGGGAAGCTGGC (SEQ ID NO: 163) oGV3493CAAATTTTTCTTTTTTTTCTGTACAGGCCAACATCAAGAAGACTATTCCAAACTTGGTC (SEQ ID NO: 164) oGV3495 TGTATGATTCGAAAGCTTCTTCACC (SEQ ID NO: 165)oGV3491 GCCAGCTTCCCCAACCCCACTAAACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTC (SEQ ID NO: 166) oGV3494GACCAAGTTTGGAATAGTCTTCTTGATGTTGGCCTGTACAGAAAAAAAAGAAAAATTTG (SEQ ID NO: 167) oGV3497 TTACTCGAGCTTGATTCTGAC (SEQ ID NO: 168)oGV2320 GGCTGTGTAGAAGTACTCGCCGATAG (SEQ ID NO: 169) oGV3496ATGTCTTCATCACTAGCAGAG (SEQ ID NO: 170) oGV0821CGGGTAATTAACGACACCCTAGAGG (SEQ ID NO: 171) oGV0706GGTTGGTATTCCAGCTGGTGTCG (SEQ ID NO: 172)

Strain Construction: The K. marxianus ALD6 gene homolog encoding the K.marxianus ALD6 protein (SEQ ID NO: 39) was deleted from parent K.marxianus strains GEVO1947 and GEVO2087 as follows, resulting in strainsGEVO6264/GEVO6265, and GEVO6270/GEVO6271 respectively.

Genomic DNA was isolated from GEVO1947 as described. Constructs weremade to integrate the E. coli hph (hygromycin resistance) cassette intothe ALD6 locus of GEVO1947 and GEVO2087 by SOE PCR as described. PCRstep #1 consisted of three reactions: the 5′ ALD6 targeting sequence,the 3′ ALD6 targeting sequence, and the hph marker. The 5′ targetingsequence was amplified from prepared GEVO1947 genomic DNA with primersoGV3490 and oGV3492. The 635 bp fragment was purified by gelelectrophoresis. The 3′ targeting sequence was amplified from preparedGEVO1947 genomic DNA with primers oGV3493 and oGV3495. The 645 bpfragment was gel purified. The P_(TEF1):hph:T_(CYC1(partial)) cassettewas amplified from pGV2701 with primers oGV3491 and oGV3494. The 1,665bp fragment was gel purified. The final SOE PCR step joined the 3products from step #1 (5′ ALD6 targeting sequence/hph/marker/3′ ALD6targeting sequence). The reaction was amplified using primers oGV3490and oGV3495. The 2,826 bp fragment was gel purified and used fortransformations of GEVO1947 and GEVO2087 as described. Medium used togrow cells for the transformation was YPD. Following the transformation,150 μL of each transformation culture was spread onto YPD platessupplemented with 0.2 g/L hygromycin. The plates were incubated at 30°C. Transformed colonies were patched for initial colony PCR screening,then single colony isolated and repatched on YPD plates supplementedwith 0.2 g/L hygromycin.

Yeast Colony PCR was used to screen for the appropriate 3′ integrationjunction, 5′ integration junction, as well as lack of the ALD6 codingregion as described. The proper 3′ integration junction was confirmedusing primers oGV3497 and oGV2320. The proper 5′ integration junctionwas confirmed using primers oGV3496 and oGV0821. Finally, deletion ofthe ALD6 internal coding region was confirmed using primers oGV3495 andoGV0706.

Fermentation: A shake flask fermentation with 2 g/L isobutyraldehyde wasperformed as described using technical triplicates of the ald6Δ strainsGEVO6264/GEVO6265 and GEVO6270/GEVO6271 and their corresponding ALD6parent strains GEVO1947 and GEVO2087.

Single colony isolated transformants of confirmed ald6Δ strains werepatched to YPD plates supplemented with 0.2 g/L hygromycin plates andparents were patched to YPD plates. Cells from the patches were used toinoculate technical triplicate 3 mL cultures of YPD. Cultures wereincubated overnight at 30° C. and 250 rpm. After overnight incubation,the OD₆₀₀ of these cultures was determined by diluting 1:40 in water.The appropriate amount of culture was added to 50 mL of YPD with 5%glucose to obtain an OD₆₀₀ of 0.1 in 250 mL baffled flasks and cultureswere incubated at 30° C. and 250 rpm. After 24 h incubation, the OD₆₀₀of these cultures was determined by diluting 1:40 in water. Theappropriate amount of culture was added to 50 mL of YPD containing 8%glucose, 200 mM MES pH 6.5, and 2 g/L isobutyraldehyde to obtain anOD₆₀₀ of 5. Fermentation cultures were incubated at 30° C. and 75 rpm inunbaffled 250 mL flasks. Unused media was collected as a media blank forLC analysis and kept at 4° C. until sample submission. At 48 h, samplesfrom each of the flasks were taken as follows. 1.5 mL of culture wasremoved into 1.5 mL Eppendorf tubes. OD₆₀₀ values were determined andsamples were prepared for LC1 analysis. Each tube was centrifuged for 10min at 14,000 rpm and the supernatant was analyzed by LC1. In additionsamples for enzyme assays were harvested after 48 h. 80 ODs of theappropriate sample were transferred into two 15 mL Falcon tube (forduplicate samples) and centrifuged at 3000×g for 5 min at 4° C. Pelletswere resuspended in 3 mL cold, sterile water and were centrifuged at5000×g for 2 min at 4° C. in a swinging bucket rotor. The water wasremoved by vacuum aspirator. The conical tubes were stored at −80° C.

Table 76 shows the isobutyrate titer after 48 h of fermentation. TheALD6 parent strain GEVO1947 produced average total and specificisobutyrate titers of 0.19 g/L and 0.013 g/L/OD, respectively. Thesetotal and specific isobutyrate titers were significantly decreased inthe ald6Δ strain GEVO6264 (0.06 g/L and 0.004 g/L/OD respectively), andalso in the ald6Δ strain GEVO6265 (0.05 g/L and 0.003 g/L/ODrespectively). The ALD6 parent strain GEVO2087 produced total andspecific isobutyrate titers of 0.15 g/L and 0.008 g/L/OD, respectively.The total and specific isobutyrate titers were significantly decreasedin the ald6Δ strain GEVO6270 (0.05 g/L and 0.003 g/L/OD), and also inthe ald6Δ strain GEVO6271 (0.08 g/L and 0.005 g/L/OD, respectively).

TABLE 76 Isobutyrate Production of ALD6 Parent Strains and ald6Δ StrainsDerived From Said ALD6 Parent Strains. Isobutyraldehyde FeedFermentation (48 hr) Parent Isobutyrate Isobutyrate Strain Strain ALD6Titer (g/L) Decrease (%) GEVO1947 + 0.19 ± 0.05 GEVO6264 GEVO1947 − 0.06± 0.02 68% GEVO6265 GEVO1947 − 0.05 ± 0.02 74% GEVO2087 + 0.15 ± 0.03GEVO6270 GEVO2087 − 0.05 ± 0.03 67% GEVO6271 GEVO2087 − 0.08 ± 0.02 47%

Example 25 Effect of ALD6 Deletion in K. lactis

The purpose of this example is to demonstrate that the deletion of ALD6in a Kluyveromyces lactis strain results in reduced isobutyraldehydeoxidation activity and isobutyrate production.

Strains, plasmids, and oligonucleotide primers disclosed in this exampleare listed in Tables 77, 78, and 79, respectively.

TABLE 77 Genotype of K. lactis Strains Disclosed in Example 25. GEVONumber Genotype GEVO1287 MATα uraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1],Kluyveromyces lactis (Dombrowski) van der Walt, teleomorph, ATCC 200826GEVO6242 MATα uraA1 trp1 leu2 lysA1 ade1 lac4-8 [pKD1]ald6Δ::P_(TEF1)-hph GEVO1830 MATα uraA1 trp1 leu2 lysA1 ade1 lac4-8[pKD1] pdc1::kan: Ec_ilvC_ΔN: Ec_ilvDΔN_coKI::Sc_LEU2 integrated}{Ll_kivD; Sc_Adh7: Km_URA3 randomly integrated} {P_(Sc) _(—) _(CUP1-1):Bs_alsS: TRP1 random integrated} GEVO6244, MATα uraA1 trp1 leu2 lysA1ade1 lac4-8 [pKD1] GEVO6245 pdc1::kan: Ec_ilvC_ΔN:Ec_ilvDΔN_coKI::Sc_LEU2 integrated} {Ll_kivD; Sc_Adh7: Km_URA3integrated} {P_(Sc) _(—) _(CUP1-1): Bs_alsS: TRP1 random integrated}ald6Δ::P_(TEF1)-hph

TABLE 78 Plasmid Disclosed in Example 25. Plasmid Name Genotype pGV2701P_(TEF1): hph, CEN, pUC ori, bla

TABLE 79 Oligonucleotide Sequences Disclosed in Example 25. PrimerSequence oGV3502 GAAACACAGTGGATTAGTGCTGTC (SEQ ID NO: 173) oGV3504GAAGAGTAAAAAAGGAGTAGAAACATTTTGAAGCTATGCTCTTTGTAATTGTTGTTGGTG (SEQ ID NO: 174) oGV3505CAAATTTTTCTTTTTTTTCTGTACAAACAGAGTCCATCCGTTTGAAACTGATTGCATGTC (SEQ ID NO: 175) oGV3507 TCAAATTCTATTATCGCGCGGG (SEQ ID NO: 176)oGV3503 CACCAACAACAATTACAAAGAGCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTC (SEQ ID NO: 177) oGV3506GACATGCAATCAGTTTCAAACGGATGGACTCTGTTTGTACAGAAAAAAAAGAAAAATTTG (SEQ ID NO: 178) oGV3509CTCCTCCGTTGCAGAACAAGGCTTTG (SEQ ID NO: 179) oGV2320GGCTGTGTAGAAGTACTCGCCGATAG (SEQ ID NO: 180) oGV3508CGGTGTTAAGTGCCAGAAATTGGTTG (SEQ ID NO: 181) oGV0821CGGGTAATTAACGACACCCTAGAGG (SEQ ID NO: 182) oGV3510CGGCGTACTCGACGTCTTGAGAAGTAG (SEQ ID NO: 183)

Strain Construction: The K. lactis ALD6 gene homolog encoding the K.lactis ALD6 protein (SEQ ID NO: 29) was deleted from parent K. lactisstrains GEVO1287 and GEVO1830 as follows, resulting in strains GEVO6242and GEVO6244/GEVO6245, respectively.

Genomic DNA was isolated from GEVO1287 as described. Constructs weremade to integrate the E. coli hph (hygromycin resistance) cassette intothe ALD6 locus of GEVO1287 and GEVO1830 by SOE PCR as described. PCRstep #1 consisted of three reactions: the 5′ ALD6 targeting sequence,the 3′ ALD6 targeting sequence, and the hph marker. The 5′ targetingsequence was amplified from prepared GEVO1287 genomic DNA with primersoGV3502 and oGV3504. The 639 bp fragment was purified by gelelectrophoresis. The 3′ targeting sequence was amplified from preparedGEVO1287 genomic DNA with primers oGV3505 and oGV3507. The 628 bpfragment was gel purified. The P_(TEF1):hph:T_(CYC1(partial)) cassettewas amplified from pGV2701 with primers oGV3503 and oGV3506. The 1,663bp fragment was gel purified. The final SOE PCR step joined the 3products from step #1 (5′ targeting sequence/hph marker/3′ targetingsequence). The reaction was amplified using primers oGV3502 and oGV3507.The 2,810 bp fragment was gel purified and used for transformations ofGEVO1287 and GEVO1830 as described. Colonies were selected forhygromycin resistance on YPD plates supplemented with 0.1 g/Lhygromycin. Yeast Colony PCR was used to screen for the appropriate 3′integration junction, 5′ integration junction, as well as lack of theALD6 coding region as described. The proper 3′ integration junction wasconfirmed using primers oGV3509 and oGV2320. The proper 5′ integrationjunction was confirmed using primers oGV3508 and oGV0821. Finally,deletion of the ALD6 internal coding region was confirmed using primersoGV3508 and oGV3510.

Fermentation: A first shake flask fermentation with 2 g/Lisobutyraldehyde in the medium was performed using technical triplicatesof the ald6Δ strain GEVO6242 and the ALD6 wild-type parent strainGEVO1287. Single colony isolated transformants of confirmed ald6Δdeletion strains were patched to YPD plates supplemented with 0.1 g/Lhygromycin plates, parent strains were patched onto YPD. Cells from thepatches were used to inoculate technical triplicate 3 mL cultures ofYPD. Cultures were incubated overnight at 30° C. and 250 rpm. Afterovernight incubation, the OD₆₀₀ of these cultures was determined bydiluting 1:40 in water. The appropriate amount of culture was added to50 mL of YPD with 5% glucose to obtain an OD₆₀₀ of 0.1 in 250 mL baffledflasks and cultures were incubated at 30° C. and 250 rpm. After 24 hincubation, the OD₆₀₀ of these cultures was determined by diluting 1:40in water. The appropriate amount of culture was added to 50 mL of YPDcontaining 8% glucose, 200 mM MES pH 6.5, and 2 g/L isobutyraldehyde toobtain an OD₆₀₀ of 5. Fermentation cultures were incubated at 30° C. and75 rpm in unbaffled 250 mL flasks. Unused media was collected as a mediablank for LC1 analysis and kept at 4° C. until sample submission. At 24h, samples from each of the flasks were taken as follows. 1.5 mL ofculture was removed into 1.5 mL Eppendorf tubes. OD₆₀₀ values weredetermined and samples were prepared for LC1 analysis as described. Eachtube was centrifuged for 10 min at 14,000 rpm and the supernatant wascollected for analysis by LC1 as described.

A second shake flask fermentation with 2 g/L isobutyraldehyde wasperformed as described using the ald6Δ deletion strainsGEVO6244/GEVO6245 and their corresponding ALD6 parent strain GEVO1830.This fermentation was sampled at 24 and 48 h as described. Table 80shows the isobutyrate titer for both of these fermentations. Isobutyratetiters are significantly decreased in the ald6Δ strains compared to theALD6 parent strains.

TABLE 80 Isobutyrate Production of ALD6 Parent Strains and ald6Δ StrainsDerived From Said ALD6 Parent Strains. Isobutyraldehyde FeedIsobutyraldehyde Feed Fermentation (24 hr) Fermentation (48 hr)Isobutyrate Isobutyrate Isobutyrate Isobutyrate Titer Decrease TiterDecrease Strain (g/L) (%) (g/L) (%) GEVO1287 0.19 ± 0.03 n.d. n.d.GEVO6242 0.12 ± 0.02 36.8% n.d. n.d. GEVO1830 0.16 ± 0.00 0.12 ± 0.01GEVO6244 0.06 ± 0.02 62.5% 0.04 ± 0.01 66.7 GEVO6245 0.07 ± 0.00 56.3%0.00 ± 0.00 ≧79.2* n.d. = not determined in this experiment *based onLOQ for isobutyrate of 0.025 g/L

Example 26 TMA29 Activity Towards 2-Aceto-2-Hydroxybutyrate

The following example illustrates that the S. cerevisiae TMA29 proteinis active towards (S)-2-acetolactate ((S)-AL) and2-aceto-2-hydroxybutyrate (AHB).

TABLE 81 Genotype of Strains Disclosed in Example 26. GEVO # GenotypeSource GEVO3527 MATα his3Δ-1 leu2Δ ATCC# 201389 lys2Δ ura3Δ (BY4742)GEVO3939 MATα his3Δ-1 leu2Δ OpenBiosystems cat# lys2Δ ura3Δ YSC1054(Yeast tma29::kan^(R) MATalpha collection)

Yeast strains GEVO3939 from which the TMA29 (YMR226C) gene was deletedand its parent GEVO3527 were each cultured in triplicate by inoculating3 mL of YPD in a 14 mL culture tube in triplicate for each strain.Cultures were started from patches on YPD agar plate for GEVO3527 and onYPD plates containing 0.2 g/L G418 for GEVO3939. The cultures wereincubated overnight at 30° C. and 250 rpm. The next day, the OD₆₀₀ ofthe overnight cultures were measured and the volume of each culture toinoculate a 50 mL culture to an OD₆₀₀ of 0.1 was calculated. Thecalculated volume of each culture was used to inoculate 50 mL of YPD ina 250 mL baffled flask and the cultures were incubated at 30° C. and 250rpm.

The cells were harvested during mid-log phase at ODs of 2.2-2.7 after 8h of growth. The cultures were transferred to pre-weighed 50 mL Falcontubes and cells were collected by centrifugation for 5 minutes at3000×g. After removal of the medium, cells were washed with 10 mL MilliQH₂0. After removal of the water, the cells were centrifuged again at3000×g for 5 minutes and the remaining water was carefully removed usinga 1 mL pipette tip. The cell pellets were weighed and then stored at−80° C. until further use.

Cell pellets were thawed on ice and resuspended in lysis buffer (10 mMsodium phosphate pH7.0, 1 mM dithiothreitol, 5% w/v glycerol) such thatthe result was a 20% cell suspension by mass. One mL of glass beads (0.5mm diameter) was added to a 1.5 mL Eppendorf tube for each sample and850 μL of cell suspension were added. Yeast cells were lysed using aRetsch MM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6×1 min eachat full speed with 1 min incubation on ice between. The tubes werecentrifuged for 10 min at 21,500×g at 4° C. and the supernatant wastransferred to a fresh tube. Extracts were held on ice until they wereassayed using the TMA29 assay as described to determine TMA29 activitytowards (R/S)-AHB and (R/S)-AL.

The specific activity of S. cerevisiae TMA29 in GEVO3527 lysates, awild-type MATa S. cerevisiae strain, for the reduction of (R/S)-AHB was10.5±0.6 mU/mg. The tma29Δ strain GEVO3939 had a specific activity of4.8±0.1 mU/mg. The wild-type GEVO3527 strain had about a 2-fold higherspecific TMA29 activity than the deletion strain.

The specific activity of S. cerevisiae TMA29 in GEVO3527 lysates, awild-type MATa S. cerevisiae strain, for the reduction of (R/S)-AL was12.3±0.2 mU/mg. The tma29Δ strain GEVO3939 had a specific activity of2.9±0.3 mU/mg. The wild-type GEVO3527 strain had about a 4-fold higherspecific TMA29 activity than the deletion strain.

General Methods for Examples 27-30

Strains, plasmids, gene/amino acid sequences, and primer sequencesdescribed in Examples 27-30 are listed in Tables 82, 83, 84, and 85,respectively.

TABLE 82 Genotype of Strains Disclosed in Examples 27-30. Genotype orreference E. coli BL21(DE3) (Lucigen Corporation, Middleton, WI) E. coliDH5α (Novagen, Gibbstown, NJ) S. cerevisiae CEN.PK2 (Euroscarf,Frankfurt, Germany)

TABLE 83 Plasmids Disclosed in Examples 27-30. Gevo No. Genotype orreference pET22(b)+ Novagen, Gibbstown, NJ pGV1102 P_(Sc) _(—)_(TEF1)-HA-tag-MCS-T_(CYC1), URA3, 2-micron, bla, pUC-ori pGV1662 P_(Sc)_(—) _(TEF1)-L. lactis kivD-T_(Sc) _(—) _(CYC1,) bla, ColE1 ori, URA3,2μ ori. pGV1947 P_(Sc) _(—) _(TEF1)-Ll_adhA-T_(Sc) _(—) _(CYC1) bla URA3pMB1 ori 2μ ori pGV1947his P_(Sc) _(—) _(TEF1)-Ll_adhA^(his6)-T_(Sc)_(—) _(CYC1) bla URA3 pMB1 ori 2μ ori pET1947 P_(T7)::Ll_adhA^(his6),bla, oripBR322, lacI pGV2274 Cloning vector containing Ll_adhA_coScsequence (synthesized by DNA2.0, Menlo Park, CA) pGV2475 P_(Sc) _(—)_(TEF1)-Ll_adhA_coSc^(28E7-his6)-T_(Sc) _(—) _(CYC1), bla, URA3, pMB1ori, 2μ ori pGV2476 P_(Sc) _(—) _(TEF1)-Ll_adhA_coSc^(his6)-T_(Sc) _(—)_(CYC1), bla, URA3, pMB1 ori, 2μ ori pGV2477 P_(Sc) _(—)_(TEF1)-Ll_adhA_coSc^(RE1-his6)-T_(Sc) _(—) _(CYC1), bla, URA3, pMB1ori, 2μ ori pGV30C11 P_(Sc) _(—)_(TEF1)-Ll_adhA_coSc^(30C11-his6)-T_(Sc) _(—) _(CYC1), bla, URA3, pMB1ori, 2μ ori

TABLE 84 Nucleic Acid and Protein Sequences Disclosed in Examples 27-30.Source Gene (SEQ ID NO) Protein (SEQ ID NO) L. lactisLl_adhA (SEQ ID NO: 184) Ll_AdhA (SEQ ID NO: 185) L. lactisLl_adhA_coSc^(his6) (SEQ ID NO: 186) Ll_AdhA^(his6) (SEQ ID NO: 187)L. lactis Ll_adhA_coSc^(28E7-his6) (SEQ ID NO: 188)Ll_AdhA^(28E7-his6) (SEQ ID NO: 189) L. lactisLl_adhA_coSc^(30C11-his6) (SEQ ID NO: 190)Ll_AdhA^(30C11-his6) (SEQ ID NO: 191) L. lactisLl_adhA_coSc^(RE1-his6) (SEQ ID NO: 192)Ll_AdhA^(RE1-his6) (SEQ ID NO: 193) L. lactisLl_adhA_coSc^(7A4-his6) (SEQ ID NO: 194)Ll_AdhA^(7A4-his6) (SEQ ID NO: 195) L. lactisLl_adhA_coSc^(4A3-his6) (SEQ ID NO: 196)Ll_AdhA^(4A3-his6) (SEQ ID NO: 197) L. lactisLl_adhA^(1H7-his6) (SEQ ID NO: 198) Ll_AdhA^(1H7-his6) (SEQ ID NO: 199)L. lactis Ll_adhA^(10F10-his6) (SEQ ID NO: 200)Ll_AdhA^(10F10-his6) (SEQ ID NO: 201) L. lactisLl_adhA^(8F11-his6) (SEQ ID NO: 202)Ll_AdhA^(8F11-his6) (SEQ ID NO: 203) L. lactisLl_adhA^(8D10-his6) (SEQ ID NO: 204)Ll_AdhA^(8D10-his6) (SEQ ID NO: 205) L. lactisLl_adhA_coSc (SEQ ID NO: 206) Ll_AdhA (SEQ ID NO: 185) L. lactisLl_adhA_coSc^(28E7) (SEQ ID NO: 207) Ll_AdhA^(28E7) (SEQ ID NO: 208)L. lactis Ll_adhA_coSc^(30C11) (SEQ ID NO: 209)Ll_AdhA^(30C11) (SEQ ID NO: 210) L. lactisLl_adhA_coSc^(RE1) (SEQ ID NO: 211) Ll_AdhA^(RE1) (SEQ ID NO: 212)L. lactis Ll_adhA_coSc^(7A4) (SEQ ID NO: 213)Ll_AdhA^(7A4) (SEQ ID NO: 214) L. lactisLl_adhA_coSc^(4A3 )(SEQ ID NO: 215) Ll_AdhA^(4A3) (SEQ ID NO: 216)L. lactis Ll_adhA^(1H7) (SEQ ID NO: 217) Ll_AdhA^(1H7) (SEQ ID NO: 218)L. lactis Ll_adhA^(10F10) (SEQ ID NO: 219)Ll_AdhA^(10F10) (SEQ ID NO: 220) L. lactisLl_adhA^(8F11) (SEQ ID NO: 221) Ll_AdhA^(8F11) (SEQ ID NO: 222)L. lactis Ll_adhA^(8D10) (SEQ ID NO: 223)Ll_AdhA^(8D10) (SEQ ID NO: 224)

TABLE 85 Primer Sequences (shown from 5′to 3′) Disclosed in Examples 27-30. Primer Name Sequence* XX7GGAGAAAACCCATATGTCGTTTAC (SEQ ID NO: 225) XX9GCAGCCGAACGCTCGAGGGCGGCCG (SEQ ID NO: 226) His_Not1_1947_revCTCGAGCGGCCGCTTAGTGGTGGTGGTGGTGGTGTTTAGTAAA ATCAA (SEQ ID NO: 227)Sal1_for GAAAGCATAGCAATCTAATCTAAGTT (SEQ ID NO: 228) adhAcoSc_Sallin_forGTTTGTCGACATGAAGGCTGCAGTTGTCCGT (SEQ ID NO: 229) adhAcoSC_Notlin_his_revTCGAGCGGCCGCTTAGTGGTGGTGGTGGTGGTGCTTCGTGAAGTCTATAACCATTCTACC (SEQ ID NO: 230) pGV1994ep_forCGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATT ACAAC (SEQ ID NO: 231)pGV1994ep_rev CTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGG (SEQ ID NO: 232)RecombADHY50_for TGCTGCCGGAGATTWCGGCAACAAGGCAGG (SEQ ID NO: 233)RecombADHY50_rev CCTGCCTTGTTGCCGWAATCTCCGGCAGCA (SEQ ID NO: 234)RecombADHL264_for ATGGTAGCCGTTGCTKTACCAAACACAGAA (SEQ ID NO: 235)RecombADHL264_rev TTCTGTGTTTGGTAMAGCAACGGCTACCAT (SEQ ID NO: 236)RecombADHI212_Y219_for GCTGATGTCAYAATTAACTCTGGTGACGTTWACCCTGTAG(SEQ ID NO: 237) RecombADHI212_Y219_revCTACAGGGTWAACGTCACCAGAGTTAATTRTGACATCAGC (SEQ ID NO: 238) NNKADHF50_forTGCTGCCGGAGATNNKGGCAACAAG (SEQ ID NO: 239) NNKADHF50_revGCCTTGTTGCCMNNATCTCCGGCAG (SEQ ID NO: 240) NNKADHR77_forGTTAGTTCTCTCNNKGTAGGTGATAG (SEQ ID NO: 241) NNKADHR77_revCACTCTATCACCTACMNNGAGAGAAC (SEQ ID NO: 242) NNKADHA108_forACATTTTGCCGAGAANNKAAAAACGC (SEQ ID NO: 243) NNKADHA108_revACCAGCGTTTTTMNNTTCTCGGCAAA (SEQ ID NO: 244) NNKADHF113_forGTCAAAAACGCTGGTNNKAGCGTTGA (SEQ ID NO: 245) NNKADHF113_revACCATCAACGCTMNNACCAGCGTTTT (SEQ ID NO: 246) NNKADHT212_forAGATAGGTGCTGATGTCNNKATTAAC (SEQ ID NO: 247) NNKADHT212_revCAGAGTTAATMNNGACATCAGCACCT (SEQ ID NO: 248) NNKADHV264_forGGTAGCCGTTGCTNNKCCAAACACAG (SEQ ID NO: 249) NNKADHV264_revATTTCTGTGTTTGGMNNAGCAACGGC (SEQ ID NO: 250) Recomb2F50Minilib_forGTTGCAGCAGGTGATTDKGGCAACAAAGCA (SEQ ID NO: 251) Recomb2F50Minilib_revTGCTTTGTTGCCMHAATCACCTGCTGCAAC (SEQ ID NO: 252) Recomb2Q77Gen5_for3TGATGTAAGCTCGCTTCAAGTTGGTGATCG (SEQ ID NO: 253) Recomb2Q77Gen5_rev4CGATCACCAACTTGAAGCGAGCTTACATCA (SEQ ID NO: 254) Recomb2R77Gen5_for5TGATGTAAGCTCGCTTCGAGTTGGTGATCG (SEQ ID NO: 255) Recomb2R77Gen5_rev6CGATCACCAACTCGAAGCGAGCTTACATCA (SEQ ID NO: 256) Recomb2S77Gen5_for7TGATGTAAGCTCGCTTTCTGTTGGTGATCG (SEQ ID NO: 257) Recomb2S77Gen5_rev8CGATCACCAACAGAAAGCGAGCTTACATCA (SEQ ID NO: 258) Recomb2Y113 Gen5_for9TTAAAAATGCAGGATATTCAGTTGATGGCG (SEQ ID NO: 259) Recomb2Y113 Gen5_rev10CGCCATCAACTGAATATCCTGCATTTTTAA (SEQ ID NO: 260) Recomb2F113 Gen5_for11TTAAAAATGCAGGATTTTCAGTTGATGGCG (SEQ ID NO: 261) Recomb2F113 Gen5_rev12CGCCATCAACTGAAAATCCTGCATTTTTAA (SEQ ID NO: 262) Recomb2G113 Gen5_for13TTAAAAATGCAGGAGGGTCAGTTGATGGCG (SEQ ID NO: 263) Recomb2G113 Gen5_rev14CGCCATCAACTGACCCTCCTGCATTTTTAA (SEQ ID NO: 264) Recomb2T212 Mini_for15GAGCTGATGTGRYAATCAATTCTGGTGATG (SEQ ID NO: 265) Recomb2T212 Mini_rev16CATCACCAGAATTGATTRYCACATCAGCTC (SEQ ID NO: 266) Recomb2V264 Mini_for17TGGTTGCTGTGGCAKTACCCAATACTGAGA (SEQ ID NO: 267) Recomb2V264 Mini_rev18TCTCAGTATTGGGTAMTGCCACAGCAACCA (SEQ ID NO: 268) *A (Adenine), G(Guanine), C (Cytosine), T (Thymine), U (Uracil), R (Purine - A or G), Y(Pyrimidine - C or T), N (Any nucleotide), W (Weak - A or T), S(Strong - G or C), M (Amino - A or C), K (Keto - G or T), B (Not A -G orC or T), H (Not G - A or C or T), D (Not C - A or G or T), and V (NotT - A or G or C)

Media and Buffers:

SC-URA: 6.7 g/L Difco™ Yeast Nitrogen Base, 14 g/L Sigma™ Synthetic

Dropout Media supplement (includes amino acids and nutrients excludinghistidine, tryptophan, and leucine), 10 g/L casamino acids, 20 g/Lglucose, 0.018 g/L adenine hemisulfate, and 0.076 g/L tryptophan.

SD-URA: Commercially available at MP Biomedicals (Irvine, Calif.).Composition: 1.7 g/L yeast nitrogen base (YNB), 5 g/L ammonium sulfate,20 g/L glucose, with casamino acids without uracil CSM-URA.

YPD (yeast peptone dextrose) media: 10 g/L yeast extract, 20 g/Lpeptone, 20 g/L glucose.

Tris-DTT: 0.39 g 1,4-dithiothreitol per 1 mL of 1 M TrisHCl, pH 8.0,filter sterilized.

Buffer A: 20 mM Tris, 20 mM imidazol, 100 mM NaCl, 10 mM MgCl₂, adjustedto pH 7.4, filter sterilized.

Buffer B: 20 mM Tris, 300 mM imidazol, 100 mM NaCl, 10 mM MgCl₂,adjusted to pH 7.4, filter sterilized.

Buffer E: 1.2 g Tris base, 92.4 g glucose, and 0.2 g MgCl₂ per 1 L ofdeionized water, adjusted to pH 7.5, filter sterilized.

Construction of pET1947: The L. lactis adhA (LI_adhA) gene was clonedout of pGV1947 using primers His_Not1_(—)1947_fwd and Sal1_rev andligated into pET22b(+), yielding plasmid pET1947.

Construction of pGV2476: Plasmid pGV2274 served as template for PCRusing forward primer adhAcoSc_Sallin_for and reverse primeradhAcoSC_Notlin_his_rev. The PCR product was purified, restrictiondigested with NotI and SalI, and ligated into pGV1662, which had beencut with NotI and SalI and purified.

Transformation of S. cerevisiae: In the evening before a plannedtransformation, a YPD culture was inoculated with a single S. cerevisiaeCEN.PK2 colony and incubated at 30° C. and 250 rpm over night. On thenext morning, a 20 mL YPD culture was started in a 250 mL Erlenmeyerflask without baffles with the overnight culture at an OD₆₀₀ of 0.1.This culture was incubated at 30° C. and 250 rpm until it reached anOD₆₀₀ of 1.3-1.5. When the culture had reached the desired OD₆₀₀, 200 μLof Tris-DTT were added, and the culture was allowed to incubate at 30°C. and 250 rpm for another 15 min. The cells were then pelleted at 4° C.and 2,500×g for 3 min. After removing the supernatant, the pellet wasresuspended in 10 mL of ice-cold buffer E and spun down again asdescribed above. Then, the cell pellet was resuspended in 1 mL ofice-cold buffer E and spun down one more time as before. After removalof the supernatant with a pipette, 200 μL of ice-cold buffer E wereadded, and the pellet was gently resuspended. The 6 μL ofinsert/backbone mixture was split in half and added to 50 μL of the cellsuspension. The DNA/cell mixtures were transferred into 0.2 cmelectroporation cuvettes (BIORAD) and electroporated without a pulsecontroller at 0.54 kV and 25 μF. Immediately, 1 mL of pre-warmed YPD wasadded, and the transformed cells were allowed to regenerate at 30° C.and 250 rpm in 15 mL round bottom culture tubes (Falcon). After 1 hour,the cells were spun down at 4° C. and 2,500×g for 3 min, and the pelletswere resuspended in 1 mL pre-warmed SD-URA media. Different amounts oftransformed cells were plated on SD-URA plates and incubated at 30° C.for 1.5 days or until the colonies were large enough to be picked withsterile toothpicks.

Plasmid Mini-Preparation of Yeast Cells: The Zymoprep™ II—Yeast PlasmidMiniprep kit (Zymo Research, Orange, Calif.) was used to prepare plasmidDNA from S. cerevisiae cells according to the manufacturer's protocolfor liquid cultures, which was slightly altered. An aliquot of 200 μL ofyeast cells was spun down at 600×g for 2 min. After decanting thesupernatant, 200 μL of Solution 1 were added to resuspend the pellets.To the samples, 3 μL of Zymolyase™ were added and the cell/enzymesuspensions were gently mixed by flicking with a finger. Afterincubating the samples for 1 hour at 37° C., Solutions 2 and 3 wereadded and mixed well after each addition. The samples were then spundown at maximum speed and 4° C. for 10 min. The following clean-up overZymo columns was performed according to the manufacturer's instructions.The plasmid DNA was eluted with 10 μL of PCR grade water. Half of thisvolume was used to transform E. coli DH5α.

Heterologous ADH expression in E. coli: Flasks (500 mL Erlenmeyer)containing 50 mL of Luria-Bertani (LB) medium (10 g tryptone, 10 g NaCl,5 g yeast extract per liter) with ampicillin (final concentration 0.1mg/mL) were inoculated to an initial OD₆₀₀ of 0.1 using 0.5 mL overnightLB_(amp) culture of a single colony carrying plasmid pET1947. The 50 mLLB expression culture was allowed to grow for 3-4 h at 250 rpm and 37°C. Protein expression was induced at OD₆₀₀ of about 1 with the additionof IPTG to a final concentration of 0.5 mM. Protein expression wasallowed to continue for 24 h at 225 rpm and 25° C. Cells were harvestedat 5300×g and 4° C. for 10 min, and then cell pellets were frozen at−20° C. until further use.

Heterologous Expression in S. cerevisiae CEN.PK2: Flasks (1000 mLErlenmeyer) filled with 100 mL of SC-URA were inoculated with 1 mLovernight culture (5 mL SC-URA inoculated with a single CEN.PK2 colony,grown at 30° C. and 250 rpm). The expression cultures were grown at 30°C. and 250 rpm for 24 hours. The cells were pelleted at 5300×g for 5min. The supernatant was discarded and the pellets were spun again. Theresidual supernatant was then taken off with a pipette. The pellets werefrozen at −20° C. until further use.

Heterologous Expression in CEN.PK2 in 96-Well Plates for High ThroughputAssays Shallow 96-well plates, 1 mL capacity per well, filled with 300μL of SC-URA were inoculated with single CEN.PK2 colonies carryingplasmids coding for LI_adhA^(his6) or variants thereof. Deep 96-wellplates, 2 mL capacity per well, filled with 600 μL of SC-URA per wellwere inoculated with 50 μL of these overnight cultures. The plates weregrown at 30° C. and 250 rpm for 24 h, and were then harvested at 5300×gfor 5 min and 4° C. and stored at −20° C.

Preparation of ADH-Containing Extracts from E. coli: E. coli cellpellets containing expressed ADH were thawed and resuspended (0.25 g wetweight/mL buffer) in buffer A. The resuspended cells were lysed bysonication for 1 min with a 50% duty cycle and pelleted at 11000×g and4° C. for 10 min. Extracts were stored at 4° C.

Preparation of ADH-Containing Extracts from S. cerevisiae CEN.PK2: S.cerevisiae CEN.PK2 cell pellets containing expressed ADH were thawed andweighed to obtain the wet weight of the pellets. Cells were thenresuspended in buffer A such that the result was a 20% cell suspensionby mass. Glass beads of 0.5 mm diameter were added to the 1000 μL-markof (0.5 mm diameter) of a 1.5 mL Eppendorf tube, before 875 μL of cellsuspension were added. Yeast cells were lysed by bead beating using aRetsch MM301 mixer mill (Retsch Inc. Newtown, Pa.), mixing 6×1 min eachat full speed with 1-min icing steps between. The tubes were centrifugedfor 10 min at 23,500×g and 4° C., and the supernatant was removed.Extracts were stored at 4° C.

Purification of ADH: The ADH was purified by IMAC (Immobilized metalaffinity chromatography) over a 1 mL Histrap High Performance (histrapHP) column pre-charged with Nickel (GE Healthcare) using an Aktapurifier FPLC system (GE Healthcare). The column was equilibrated withfour column volumes (cv) of buffer A. After injecting the crude extractsonto the column, the column was washed with buffer A for 2 cvs, followedby a linear gradient to 100% elution buffer B for 15 cvs and collectedin 96-well plates. The fractions containing the protein were pooled andat stored at 4° C.

ADH Cuvette Assay: ADH activity was assayed kinetically by monitoringthe decrease in NADH concentration by measuring the absorbance at 340nm. A reaction buffer was prepared containing 100 mM Tris/HCl pH 7.0, 1mM DTT, 11 mM isobutyraldehyde, and 200 μM NADH. The reaction wasinitiated by addition of 100 μL of crude extract or purified protein inan appropriate dilution to 900 μL of the reaction buffer.

ADH Microtiter Plate Activity Assay: The activity measurement inmicrotiter plates is a downscaled cuvette assay. The total volume was100 μL. Ten μL of crude lysates or purified enzyme, appropriatelydiluted, were placed in assay plates. The reaction buffer was preparedas described above (isobutyraldehyde substrate only) and 90 μL thereofwere added to the enzyme solutions in the plates. The consumption ofNADH was recorded at 340 nm in an infinite M200 plate reader (TECANTrading AG, Switzerland).

ADH High-Throughput Activity Assay: Frozen yeast cell pellets in 96-wellplates were thawed at room temperature for 20 min, and then 100 μL ofY-Per (Pierce, Cat#78990) were added. Plates were vortexed briefly toresuspend the cell pellets. After a 60-min incubation period at roomtemperature and 130 rpm, 300 μL of 100 mM Tris-HCl (pH 7.0) were addedto the plates to dilute the crude extract. Following a centrifugationstep at 5,300×g and 4° C. for 10 min, 40 μL of the resulting crudeextract were transferred into assay plates (flat bottom, Rainin) using aliquid handling robot. The assay plates were briefly spun down at 4,000rpm and room temperature. Twelve mL assay buffer per plate were prepared(100 mM Tris-HCl, pH 7.0, 1 mM, 0.5 mM, 0.25 mM or 0.125 mMisobutyraldehyde, 1 mM DTT, 200 μM NADH) and 100 μL thereof were addedto each well to start the reaction. The depletion of NADH was monitoredat 340 nm in an infinite M200 plate reader (TECAN Trading AG,Switzerland) over 2 min.

Determination of Specific Activity Based on Data Obtained from theActivity Assays: The protein concentrations of samples containingheterologously expressed L. lactis AdhA, such as crude extract andpurified proteins, were measured using the Quick Start™ Bradford Kit(Bio-Rad, Hercules, Calif.) following the manufacturer's instructions.One unit of enzyme activity (1 U) is defined as the amount of enzymethat catalyzes the conversion of one micromole of substrate per minuteunder the specified conditions of the assay method.

Thermostability Measurements: T₅₀ values (temperature, at which 50% ofthe enzyme activity is retained after an incubation time of 15 min) ofthe parent LI_adhA and variants thereof were measured to obtainthermostability data. Thirty μL aliquots of purified enzyme weretransferred into PCR tubes. Each tube was assigned to a specificincubation temperature, which corresponded to a slot on the block of aMastercycler®ep PCR machine (Eppendorf, Hamburg, Germany) programmedwith a gradient covering a 20° C.-temperature range. The tubes wereincubated for 15 min in their slots. Then, the reaction was quenched onice. The residual activity was determined with the ADH microtiter plateactivity assay as described above.

Use of His-Tags for Purification: In each of the examples describedbelow, reference is made to an ADH enzyme comprising a his-tag. As isunderstood in the art, such his-tags facilitate protein purification. Aswould be understood by one skilled in the art equipped with the presentdisclosure, ADH enzymes lacking said his-tags are equally or bettersuited for the conversion of isobutyraldehyde to isobutanol. Examples ofthe modified ADH enzymes described herein which lack thepurification-enabling his-tags are found in SEQ ID NOs: 206-224.

Example 27 Directed Evolution Via Random Mutagenesis

The following example illustrates a method for improving kineticproperties of an ADH and also describes the kinetic properties of suchimproved ADH enzymes.

Plasmid pGV2476, a derivative of plasmid pGV1662, carrying theLI_adhA_coSc^(his6) gene served as template for error prone PCR usingforward primer pGV1994ep_for and reverse primer pGV1994_rev. Theseprimers are specific to the backbone pGV1662 and bind 50 bp upstream anddownstream of the ADH insert to create an overlap for homologousrecombination in yeast. The compositions of the three error prone PCRreactions are summarized in Table 86. The temperature profile was thefollowing: 95° C. 3 min initial denaturation, 95° C. 30s denaturation,55° C. 30s annealing, 72° C. 2 min elongation, 25 cycles, 5 min finalelongation at 72° C.

TABLE 86 PCR Conditions for the Error Prone Libraries. final MnCl₂ conc.[μM] 100 200 300 Template [ng] 2 2 2 primer forward [μM] 0.2 0.2 0.2primer reverse [μM] 0.2 0.2 0.2 dNTP's [μM] 400 400 400 Taq buffer (10xstock) [μL] 10 10 10 MgCl₂ [μM] 7 7 7 Taq polymerase [U] 8 8 8 MnCl₂ (1mM stock) [μM] 100 200 300 PCR grade water [μL] 41.4 31.4 21.4

The PCR products were checked on a 1% analytical TAE agarose gel, DpnIdigested for 1 h at 37° C. to remove traces of template DNA, and thencleaned up using a 1% preparative TAE agarose gel. The agarose piecescontaining the PCR products were cleaned using Freeze ‘n’ Squeeze tubes(BIORAD, Hercules, Calif.; catalog #732-6166) followed by pellet paintprocedure (Novagen, catalog #69049-3) according to manufacturers'protocols. In the meantime, plasmid pGV1662 was restriction digestedwith NotI and SalI before running out the digestion mixture on anagarose gel and pellet painting. Plasmid and insert, 500 ng each, weremixed together, precipitated with pellet paint, resuspended in 6 μL ofPCR grade water, and used to transform electrocompetent S. cerevisiaecells as described in General Methods.

A total of 88 clones from each of the 100, 200, and 300 μM MnCl₂libraries were picked into 96-well plates along with four clonescontaining parent plasmid pGV2476 and three clones containing pGV1102 asno-ADH control. One well was left empty and served as a sterilitycontrol. After screening these libraries as described under GeneralMethods (Heterologous expression in CEN.PK2 in 96-well plates for highthroughput assays, ADH high-throughput activity assay), the 300 μMlibrary was chosen and an additional 4,000 clones were screened in thesame way. A total of 24 variants had a more than 1.5-fold improvementcompared to wild type and were chosen for a re-screen in triplicate. Thetop ten variants thereof were grown and expressed in 100 mL cultures asdescribed under General Methods (Heterologous expression in S.cerevisiae CEN.PK2), and their specific activities in crude yeastextracts were determined as described under General Methods (ADHmicrotiter plate assay). Two variants, LI_AdhA^(28E7-his6) andLI_AdhA^(30C11-his6) exhibited a more than 2-fold improvement inactivity (0.3 and 0.25 U/mg total lysate protein, respectively) comparedto the wild-type enzyme LI_AdhA^(his6) (0.1 U/mg total lysate protein)and were characterized in greater detail.

Plasmid DNA from these two variants was extracted as described underGeneral Methods (Plasmid mini-preparation out of yeast cells) andsubjected to DNA sequencing (Laragen, Los Angeles, Calif.), whichrevealed two mutations per variant as listed in Table 87. Two of thesemutations (Y50F and L264V) are localized in close proximity to theactive site which is a gap between the substrate binding domain (cyan)and the cofactor binding domain (green). Mutations I212T and N219Y arelocated on the surface of the cofactor binding domain (as shown in FIG.17). In order to highlight the location of the cofactor binding sitemutations, FIG. 17 entails two views on the structure alignment.

TABLE 87 List of Mutations Found in Two Improved Variants of the FirstError Prone Library (Generation 1). Variant MutationsLl_adhA^(28E7-his6) N219Y, L264V Ll_adhA^(30C11-his6) Y50F, I212T

The two enzyme variants, LI_AdhA^(28E7-his6) and LI_AdhA^(30C11-his6),were expressed from plasmids pGV2475 and pGV30C11, respectively onlarger scale (100 mL cultures each), purified, and characterized ingreater detail as described under General Methods (Heterologousexpression in S. cerevisiae CEN.PK2, Preparation of ADH-containingextracts from S. cerevisiae CEN.PK2, Purification of ADH). The wild-typeLI_AdhA^(his6) enzyme was expressed from plasmid pGV2476 and purified inthe same way. The enzymes were characterized for the kinetic propertiesas described under General Methods (ADH cuvette assay). Table 88 showsthe kinetic parameters measured with isobutyraldehyde and NADH. Adecreased K_(M)-value was observed for both variants, while the k_(cat)was only improved for LI_AdhA^(28E7).

TABLE 88 Kinetic Parameters of the Two Variants (Generation 1) onIsobutyraldehyde Compared to the Parental Enzyme. Crude Purified ProteinLysate K_(M) k_(cat) k_(cat)/K_(M) Variant U/mg [mM] U/mg [s⁻¹][M⁻¹*s⁻¹] Ll_AdhA^(his6) 0.28 ± 0.08 11.7 ± 0.6  25 ± 0.2 30 2,800Ll_AdhA^(28E7-his6) 0.74 ± 0.15 2.7 ± 0.2 43 ± 2  60 21,000Ll_AdhA^(30C11-his6) 0.46 ± 0.2  3.9 ± 0.1 60 ± 0.2 80 20,000

The thermostability of the wild-type enzyme and the two variants wasdetermined as described under General Methods (Thermostabilitymeasurements). The mutations found had a positive impact on thestability of the variants in addition to the beneficial effects on theircatalytic efficiency. Table 89 summarizes the T₅₀s measured for theparent and the variants.

TABLE 89 Summary of the T₅₀s of the Parent Enzyme and the Variants.Variant T₅₀ [° C.], 15 min Ll_AdhA^(his6) 54.4 ± 0.5 Ll_AdhA^(28E7-his6)62.3 ± 0.3 Ll_AdhA^(30C11-his6) 57.6 ± 0.6

Example 28 Directed Evolution Via Recombination

The following example illustrates a method for improving kineticproperties of an ADH and also illustrates the kinetic properties of suchimproved ADH enzymes.

A second gene library (Generation 2) was constructed to recombinebeneficial mutations found in the first error prone library and thewild-type residue at each of these sites (Table 90).

TABLE 90 Amino Acid Mutations Included in the Recombination Library.Amino Acid Total # (including Position Wild-type Mutations wild-type) 50Y F 2 212 I T 2 219 Y N 2 264 L V 2

Four PCR fragments were generated using primers RecombADHY50_rev andpGV1994ep_for (fragment 1), RecombADHY50_for and RecombADHI212_Y219_rev(fragment 2), RecombADHI212_Y219_for and RecombADHL264_rev (fragment 3),and RecombADHL264_rev and pGV1994ep_rev (fragment 4). The fragments wereanalyzed on an analytical 1% TAE gel, DpnI digested, separated on a 1%preparative TAE agarose gel, Freeze‘n’Squeeze (BIORAD) treated, andfinally pellet painted (Novagen). The clean fragments served as templatefor the assembly PCR. After the successful assembly PCR, the PCRproducts were treated as described in Example 27, mixed with pGV1662backbone as described in Example 27, and the mixture was used totransform S. cerevisiae as described in General Methods for Examples27-30. Eighty single clones of the recombination library were picked andcompared in a high-throughput screen to the wild type and the twovariants found in the error prone library.

A total of 80 single clones were picked into a 96-well plate along withthe original parent and the two improved variants. After screening therecombination plate, as described under General Methods (Heterologousexpression in CEN.PK2 in 96-well plates for high throughput assays, ADHhigh-throughput activity assay), twelve variants, all exhibiting atleast two-fold higher activity compared to either parentLI_AdhA^(28E7-his6) or LI_AdhA^(30C11-his6) were grown and expressed in100 mL cultures as described under General Methods (Heterologousexpression in S. cerevisiae CEN.PK2), and their activities in crudeyeast extracts were determined as described under General Methods (ADHmicrotiter plate assay). Two variants had very similar specific activityin crude extract. LI_AdhA^(RE1) was chosen for further modifications, asits activity was 40% better than LI_AdhA^(28E7-his6) and 64% better thanLI_AdhA^(30C11-his6).

Plasmid DNA from this variant was extracted as described under GeneralMethods (Plasmid mini-preparation out of yeast cells) and subjected toDNA sequencing (Laragen, Los Angeles, Calif.), which revealed thatmutations Y50F, I212T, and L264V (found in LI_AdhA^(RE1)) contributed tothe observed improvements, whereas the mutation at position 219 wasdeleterious for the activity of the variants and was not found in any ofthe improved variants of the recombination library.

The variant LI_AdhA^(RE1-his6) was expressed from plasmid pGV2477, onlarger scale (100 mL cultures each), purified, and characterized ingreater detail as described under General Methods (Heterologousexpression in S. cerevisiae CEN.PK2, Preparation of ADH-containingextracts from S. cerevisiae CEN.PK2, Purification of ADH). The wild-typeLI_AdhA^(his6) enzyme was expressed from plasmid pGV2476 and purified inthe same way. The enzymes were characterized for the kinetic propertiesas described under General Methods (ADH cuvette assay). Table 91 showsthe kinetic parameters measured with isobutyraldehyde and NADH. Comparedto LI_AdhA^(his6), LI_AdhA^(28E7-his6), LI_AdhA^(30C11-his6) a decreasedK_(M) and an increased k_(cat) was observed for LI_AdhA^(RE1-his6).

TABLE 91 Biochemical Properties of Ll_AdhA^(RE1) as Measured onIsobutyraldehyde. Crude Purified Protein Lysate K_(M) k_(cat)k_(cat)/K_(M) Variant U/mg [mM] U/mg [s⁻¹] [M⁻¹*s⁻¹] Ll_AdhA^(his6) 0.28± 0.08 11.7 ± 0.6  25 ± 0.2 30 2,800 Ll_AdhA^(28E7-his6) 0.74 ± 0.15 2.7± 0.2 43 ± 2  60 21,000 Ll_AdhA^(30C11-his6) 0.46 ± 0.2  3.9 ± 0.1 60 ±0.2 80 20,000 Ll_AdhA^(RE1-his6) 1.15 ± 0.2  1.7 ± 0.0 105 ± 1   14082,000

Variant LI_adhA^(RE1-his6), exhibited a T₅₀ value of 61.6±0.1° C. whichis 5 degrees higher than the T₅₀ of the wt and roughly 1 degree lowerthan the most stable parent of the recombination round,LI_AdhA^(28E7-his6).

Example 29 Directed Evolution of the L. lactis AdhA Via RandomMutagenesis, Site Saturation Mutagenesis, and Recombination

The following example illustrates a method for improving kineticproperties of an ADH and also describes the kinetic properties of suchimproved ADH enzymes.

The LI_adhA^(RE1-his6) gene served as template for a second round oferror prone PCR and screening (Generation 3). The screening assayutilized 0.125 mM isobutyraldehyde. About 3,000 clones of a librarygenerated using error prone PCR with 200 μM MnCl₂ according to Example 1above were expressed and screened in a high throughput fashion. Severalhits were chosen for a rescreen in triplicate and two variants,LI_AdhA^(7A4-his6) and LI_AdhA^(4A3-his6), were identified with improvedactivity. The mutations of these variants are depicted in Table 92.

TABLE 92 List of Mutations Accumulated in Generation 3 VariantsLl_AdhA^(7A4-his6) and Ll_AdhA^(4A3-his6). Variant MutationsLl_AdhA^(7A4-his6) Y50F, I212T, L264V, Q77R, V108A Ll_AdhA^(4A3-his6)Y50F, I212T, L264V, Y113F

The specific activities (U/mg) in lysates of LI_AdhA^(7A4-his6) andLI_AdhA^(4A3-his6), as well as the parents, were measured in biologicaltriplicates at pH 7.0 (Table 93).

TABLE 93 Biochemical Properties of Ll_AdhA^(7A4-his6) andLl_AdhA^(4A3-his6) at pH 7.0. Crude Purified Protein Lysate K_(M)k_(cat) k_(cat)/K_(M) Variant U/mg [mM] U/mg [s⁻¹] [M⁻¹*s⁻¹]Ll_AdhA^(7A4-his6) 1.14 ± 0.1 1.2 ± 0.2 88.8 ± 2.9 117 94,000Ll_AdhA^(4A3-his6) 1.36 ± 0.1 0.9 ± 0.1  70 ± 2.9 95 100,000

The T₅₀ values of LI_AdhA^(7A4-his6) (59.4° C.) and LI_AdhA^(4A3-his6)(57.6° C.) were both higher than LI_AdhA^(his6) and lower thanLI_AdhA^(RE1-his6).

After two rounds of error prone PCR and one round of recombination,site-saturation mutagenesis was performed at each of the six sites,generating six libraries (library 50, 77, 108, 113, 212, and 264). Theoriginal parent, LI_AdhA^(his6), was used as template for each NNKfragment. Two fragments for each library were amplified using primerslisted in Table 4 (pGV1994ep_for and NNKADHF50_rev for fragment 1 oflibrary 50, NNKADHF50_for and pGV1994ep_rev for fragment 2 of library50; pGV1994ep_for and NNKADHR77_rev for fragment 1 of library 77,NNKADHR77_for and pGV1994ep_rev for fragment 2 of library 77;pGV1994ep_for and NNKADHA108_rev for fragment 1 of library 108,NNKADHA108_for and pGV1994ep_rev for fragment 2 of library 108;pGV1994ep_for and NNKADHF113_rev for fragment 1 of library 113,NNKADHF113_for and pGV1994ep_rev for fragment 2 of library 113;pGV1994ep_for and NNKADHT212_rev for fragment 1 of library 212,NNKADHT212_for and pGV1994ep_rev for fragment 2 of library 212;pGV1994ep_for and NNKADHV264_rev for fragment 1 of library 264,NNKADHV264_for and pGV1994ep_rev for fragment 2 of library 264), andwere then used as templates for assembly PCR. The assembly PCR productswere treated as described before to generate the NNK libraries in yeast.Ninety clones were picked for each NNK library, and screened separately.After rescreening, nine clones from six libraries were mini-prepped fromyeast, the plasmids were used to transform E. coli, and the resultingplasmids were sequenced. Their lysate activities and sequencing resultsare summarized in Table 94.

TABLE 94 Summary of Site Saturation Mutagenesis (Generation 4). MutationExemplary Position of Found in Mutations NNK NNK Found in Mutations forLibraries Variant U/mg in Lysate Library NNK Library Recombination —Ll_adhA^(his6) 0.28 ± 0.08 — Ll_AdhA^(RE1) 1.15 ± 0.20 50 1G4 0.78 ±0.02 Y50W F, W C, L, F, W, Y, X 77 2G3 0.42 ± 0.00 Q77S R, S Q, R, S 772H2 0.43 ± 0.00 — — — 108 3D10 0.61 ± 0.01 V108A — — 108 3D12 0.53 ±0.07 V108S — — 113 4A3b 0.38 ± 0.05 Y113G F, G Y, F, G 113 4E6 0.30 ±0.04 Y113G — — 212 5D2 0.92 ± 0.02 I212V T, V A, I, T, V 264 6E12 0.38 ±0.07 L264V V I, V

A variety of mutations found in the site saturation mutagenesislibraries were recombined in a combinatorial fashion using SOE PCR andthe library was constructed using non-codon optimized parent,pGV1947his. The primers described in Table 85 allowed for wild-typesequence at the six targeted sites as well. Six fragments were generatedusing Recomb2F50Minilib_rev and pGV1994ep_for (fragment 1),Recomb2F50Minilib_for and mix of Recomb2Q77Gen5_rev4,Recomb2R77Gen5_rev6 and Recomb2S77Gen5_rev8 (fragment 2), mix ofRecomb2Q77Gen5_for 3, Recomb2R77Gen5_for 5 and Recomb2S77Gen5_for 7 andmix of Recomb2Y113 Gen5_rev10, Recomb2F113 Gen5_rev12 and Recomb2G113Gen5_rev14 (fragment 3), mix of Recomb2Y113 Gen5_for 9, Recomb2F113Gen5_for 11 and Recomb2G113 Gen5_for 13 and Recomb2T212 Mini_rev16(fragment 4), Recomb2T212 Mini_for 15 and Recomb2V264 Mini_rev18(fragment 5), and Recomb2V264 Mini_for 17 and pGV1994ep_rev (fragment6). The fragment PCRs were analyzed on an analytical 1% TAE gel andthen, the products were DpnI digested for 1 h at 37° C., separated on a1% preparative TAE agarose gel, Freeze‘n’Squeeze (BIORAD) treated, andfinally pellet painted (Novagen). The clean fragments served as templatefor the assembly PCR. After the successful assembly PCR, homologousrecombination (as described above) was used to create the library. Overa thousand individual clones were screened using an isobutyraldehydeconcentration of 0.125 mM. A rescreening plate was compiled consistingof the top 60 variants and assayed with 0.125 mM isobutyraldehyde.

Ten variants were chosen for expression in 100 mL SC-URA medium todetermine their specific activities in lysate. Four of them weresequenced (See Table 95 for mutations), purified, and characterized ingreater detail (Table 96). The new variants showed similar specificactivities in lysate as LI_AdhA^(RE1). Notably, variant 4A3 stood out asan enzyme with the high specific activity.

TABLE 95 List of Mutations in Variants from Generation 5. VariantMutations Ll_AdhA^(1H7-his6) Y50F, I212A, L264V, Y113FLl_AdhA^(10F10-his6) Y50F, I212T, L264V, Q77S, Y113F Ll_AdhA^(8F11-his6)Y50F, I212A, L264V, Q77R, Y113F Ll_AdhA^(8D10-his6) Y50F, I212V, L264V,Q77S, Y113F

TABLE 96 Biochemical Properties of Variants from Generation 5. CrudePurified Protein Lysate K_(M) k_(cat) k_(cat)/K_(M) Variant U/mg [mM]U/mg [s⁻¹] [M⁻¹*s⁻¹] Ll_AdhA^(1H7-his6) 1.12 ± 0.11 39.9 ± 1.7Ll_AdhA^(10F10-his6) 1.15 ± 0.17  75.4 ± 12.3 Ll_AdhA^(8F11-his6) 1.09 ±0.13 0.8 ± 0.2 41.7 ± 0.2 55 68233 Ll_AdhA^(8D10-his6) 1.05 ± 0.08 58.8± 4.5

Example 30 Engineering of Homologous ADH Enzymes

The following example illustrates how additional ADH enzymes areidentified and engineered for improving kinetic properties of additionalADH enzymes.

Enzymes homologous to the L. lactis AdhA were identified through BlastPsearches of publicly available databases using amino acid sequence of L.lactis AdhA (SEQ ID NO: 185) with the following search parameters:Expect threshold=10, word size=3, matrix=Blosum62, gap opening=11, gapextension=1. The top hundred hits, representing homologues with aboutgreater than about 60% sequence identity were selected and are listed inTable 97. The sequences were aligned using the multiple sequencealignment tool AlignX, a component of Vector NTI Advance 10.3.1 with thefollowing parameters: Gap opening pentalty=10, gap extensionpenalty=0.05, gap separation penalty range=8. The multiple sequencealignment showed very high levels of conservation amongst the residuescorresponding to (a) tyrosine 50 of the L. lactis AdhA (SEQ ID NO: 185);(b) glutamine 77 of the L. lactis AdhA (SEQ ID NO: 185); (c) valine 108of the L. lactis AdhA (SEQ ID NO: 185); (d) tyrosine 113 of the L.lactis AdhA (SEQ ID NO: 185); (e) isoleucine 212 of the L. lactis AdhA(SEQ ID NO: 185); and (f) leucine 264 of the L. lactis AdhA (SEQ ID NO:185), wherein AdhA (SEQ ID NO: 185) is encoded by the L. lactis alcoholdehydrogenase (ADH) gene adhA (SEQ ID NO: 184) or a codon-optimizedversion thereof (SEQ ID NO: 206).

TABLE 97 Homologous Enzymes with >60% Sequence Identity to L. lactisAdhA % Seq. Accession Description Identity E value Total ScoreYP_003354381.1 alcohol dehydrogenase 1 [Lactococcus lactis subsp. 100 0684 lactis KF147] NP_267964.1 alcohol dehydrogenase [Lactococcus lactissubsp. 99 0 681 lactis Il1403] YP_001033251.1 alcohol dehydrogenase[Lactococcus lactis subsp. 95 0 659 cremoris MG1363] YP_811585.1 alcoholdehydrogenase [Lactococcus lactis subsp. 95 0 658 cremoris SK11]ZP_07367864.1 alcohol dehydrogenase [Pediococcus acidilactici DSM 692.00E−129 466 20284] YP_794451.1 alcohol dehydrogenase [Lactobacillusbrevis ATCC 66 1.00E−127 460 367] ZP_06197454.1 alcohol dehydrogenase[Pediococcus acidilactici 7_4] 69 8.00E−124 447 YP_001374103.1 alcoholdehydrogenase [Bacillus cereus subsp. 65 1.00E−123 447 cytotoxis NVH391-98] ZP_00741101.1 Alcohol dehydrogenase [Bacillus thuringiensisserovar 64 9.00E−123 444 israelensis ATCC 35646] ZP_04431756.1 Alcoholdehydrogenase GroES domain protein 63 1.00E−122 444 [Bacillus coagulans36D1] ZP_04101989.1 Alcohol dehydrogenase 1 [Bacillus thuringiensis 641.00E−122 443 serovar berliner ATCC 10792] ZP_03943574.1 alcoholdehydrogenase [Lactobacillus buchneri ATCC 62 2.00E−122 443 11577]YP_002338331.1 alcohol dehydrogenase [Bacillus cereus AH187] 642.00E−122 443 ZP_04145518.1 Alcohol dehydrogenase 1 [Bacillusthuringiensis 64 2.00E−122 443 serovar tochigiensis BGSC 4Y1]ZP_00236660.1 alcohol dehydrogenase, propanol-preferring [Bacillus 642.00E−122 443 cereus G9241] ZP_03954717.1 alcohol dehydrogenase[Lactobacillus hilgardii ATCC 62 2.00E−122 443 8290] ZP_06011170.1alcohol dehydrogenase 1 [Leptotrichia goodfellowii 65 2.00E−122 442F0264] ZP_07537679.1 Alcohol dehydrogenase zinc-binding domain protein63 2.00E−122 442 [Actinobacillus pleuropneumoniae serovar 9 str.CVJ13261] NP_844655.1 alcohol dehydrogenase [Bacillus anthracis str.Ames] 64 3.00E−122 442 ZP_04071880.1 Alcohol dehydrogenase 1 [Bacillusthuringiensis IBL 64 3.00E−122 442 200] YP_001844344.1 alcoholdehydrogenase [Lactobacillus fermentum IFO 64 3.00E−122 442 3956]ZP_04227732.1 Alcohol dehydrogenase 1 [Bacillus cereus Rock3-29] 644.00E−122 442 YP_002529920.1 alcohol dehydrogenase [Bacillus cereus Q1]64 4.00E−122 442 ZP_03107320.1 putative alcohol dehydrogenase,zinc-containing 64 5.00E−122 441 [Bacillus cereus NVH0597-99]YP_001644942.1 alcohol dehydrogenase [Bacillus weihenstephanensis 645.00E−122 441 KBAB4] ZP_03940565.1 alcohol dehydrogenase [Lactobacillusbrevis subsp. 62 5.00E−122 441 gravesensis ATCC 27305] ZP_05863633.1alcohol dehydrogenase [Lactobacillus fermentum 28- 64 5.00E−122 4413-CHN] ZP_04174477.1 Alcohol dehydrogenase 1 [Bacillus cereus AH1273] 645.00E−122 441 ZP_04168725.1 Alcohol dehydrogenase 1 [Bacillus mycoidesDSM 64 5.00E−122 441 2048] ZP_03945523.1 alcohol dehydrogenase[Lactobacillus fermentum 64 5.00E−122 441 ATCC 14931] YP_894846.1alcohol dehydrogenase [Bacillus thuringiensis str. Al 64 6.00E−122 441Hakam] ZP_04273257.1 Alcohol dehydrogenase 1 [Bacillus cereus BDRD- 646.00E−122 441 ST24] ZP_07337905.1 alcohol dehydrogenase [Actinobacillus63 6.00E−122 441 pleuropneumoniae serovar 2 str. 4226] YP_795183.1alcohol dehydrogenase [Lactobacillus brevis ATCC 64 6.00E−122 441 367]ZP_03234447.1 putative alcohol dehydrogenase, zinc-containing 647.00E−122 441 [Bacillus cereus H3081.97] ZP_00134308.2 COG1064:Zn-dependent alcohol dehydrogenases 63 7.00E−122 441 [Actinobacilluspleuropneumoniae serovar 1 str. 4074] NP_831985.1 alcohol dehydrogenase[Bacillus cereus ATCC 14579] 64 7.00E−122 441 NP_978607.1 alcoholdehydrogenase [Bacillus cereus ATCC 10987] 65 7.00E−122 441YP_002366965.1 alcohol dehydrogenase [Bacillus cereus B4264] 648.00E−122 441 ZP_04283955.1 Alcohol dehydrogenase 1 [Bacillus cereusATCC 64 8.00E−122 441 4342] ZP_04218185.1 Alcohol dehydrogenase 1[Bacillus cereus Rock3-44] 64 1.00E−121 441 ZP_04186048.1 Alcoholdehydrogenase 1 [Bacillus cereus AH1271] 64 1.00E−121 441 ZP_07542038.1Alcohol dehydrogenase zinc-binding domain protein 63 1.00E−121 440[Actinobacillus pleuropneumoniae serovar 11 str. 56153] YP_003664530.1alcohol dehydrogenase [Bacillus thuringiensis 64 1.00E−121 440 BMB171]ZP_04222478.1 Alcohol dehydrogenase 1 [Bacillus cereus Rock3-42] 641.00E−121 440 ZP_04305999.1 Alcohol dehydrogenase 1 [Bacillus cereus172560W] 64 1.00E−121 440 ZP_04317351.1 Alcohol dehydrogenase 1[Bacillus cereus ATCC 64 2.00E−121 440 10876] YP_001035842.1 alcoholdehydrogenase [Streptococcus sanguinis 66 2.00E−121 439 SK36]ZP_07336540.1 alcohol dehydrogenase [Actinobacillus 63 2.00E−121 439pleuropneumoniae serovar 6 str. Femo] ZP_03232573.1 putative alcoholdehydrogenase, zinc-containing 64 2.00E−121 439 [Bacillus cereus AH1134]ZP_04084316.1 Alcohol dehydrogenase 1 [Bacillus thuringiensis 642.00E−121 439 serovar huazhongensis BGSC 4BD1] YP_036379.1 alcoholdehydrogenase [Bacillus thuringiensis serovar 64 2.00E−121 439 konkukianstr. 97-27] ZP_03713785.1 hypothetical protein EIKCOROL_01470 [Eikenella64 3.00E−121 439 corrodens ATCC 23834] ZP_04300512.1 Alcoholdehydrogenase 1 [Bacillus cereus MM3] 64 3.00E−121 439 ZP_04261933.1Alcohol dehydrogenase 1 [Bacillus cereus BDRD- 64 3.00E−121 439 ST196]ZP_04197309.1 Alcohol dehydrogenase 1 [Bacillus cereus AH603] 643.00E−121 439 ZP_04289229.1 Alcohol dehydrogenase 1 [Bacillus cereusR309803] 64 6.00E−121 438 YP_001449881.1 alcohol dehydrogenase[Streptococcus gordonii str. 64 1.00E−120 437 Challis substr. CH1]YP_002886170.1 Alcohol dehydrogenase zinc-binding domain protein 641.00E−120 437 [Exiguobacterium sp. AT1b] ZP_03072955.1 Alcoholdehydrogenase GroES domain protein 63 3.00E−120 435 [Lactobacillusreuteri 100-23] ZP_01817011.1 alcohol dehydrogenase, zinc-containing 655.00E−120 435 [Streptococcus pneumoniae SP3-BS71] ZP_03974464.1 alcoholdehydrogenase [Lactobacillus reuteri CF48- 63 6.00E−120 434 3A]ZP_01824429.1 alcohol dehydrogenase, zinc-containing 65 8.00E−120 434[Streptococcus pneumoniae SP11-BS70] YP_002735395.1 alcoholdehydrogenase [Streptococcus pneumoniae 65 8.00E−120 434 JJA] CAQ49114.1alcohol dehydrogenase, propanol-preferring 63 1.00E−119 434[Staphylococcus aureus subsp. aureus ST398] ZP_01819490.1 alcoholdehydrogenase, zinc-containing 65 1.00E−119 434 [Streptococcuspneumoniae SP6-BS73] ZP_01832462.1 alcohol dehydrogenase,zinc-containing 65 1.00E−119 434 [Streptococcus pneumoniae SP19-BS75]ZP_01834441.1 alcohol dehydrogenase, zinc-containing 65 1.00E−119 433[Streptococcus pneumoniae SP23-BS72] ZP_07644167.1 alcoholdehydrogenase, propanol-preferring 65 1.00E−119 433 [Streptococcus mitisNCTC 12261] NP_344823.1 alcohol dehydrogenase [Streptococcus pneumoniae65 1.00E−119 433 TIGR4] YP_003878532.1 alcohol dehydrogenase,propanol-preferring 65 1.00E−119 433 [Streptococcus pneumoniae 670-6B]YP_001272079.1 alcohol dehydrogenase [Lactobacillus reuteri DSM 631.00E−119 433 20016] ZP_03960239.1 alcohol dehydrogenase [Lactobacillusvaginalis ATCC 62 1.00E−119 433 49540] ZP_07646288.1 alcoholdehydrogenase [Streptococcus mitis SK564] 65 2.00E−119 433YP_002739644.1 alcohol dehydrogenase [Streptococcus pneumoniae 652.00E−119 433 70585] YP_002737553.1 alcohol dehydrogenase [Streptococcuspneumoniae 65 2.00E−119 433 P1031] YP_002741829.1 alcohol dehydrogenase[Streptococcus pneumoniae 65 2.00E−119 433 Taiwan19F-14] ZP_05689169.1alcohol dehydrogenase GroES domain-containing 63 2.00E−119 432 protein[Staphylococcus aureus A9299] ZP_07642509.1 alcohol dehydrogenase[Streptococcus mitis SK597] 65 2.00E−119 432 ZP_02718124.1 alcoholdehydrogenase, propanol-preferring 65 2.00E−119 432 [Streptococcuspneumoniae CDC3059-06] ZP_07647302.1 alcohol dehydrogenase familyprotein [Streptococcus 65 2.00E−119 432 mitis SK321] NP_371129.1 alcoholdehydrogenase [Staphylococcus aureus 62 3.00E−119 432 subsp. aureusMu50] NP_645385.1 alcohol dehydrogenase [Staphylococcus aureus 623.00E−119 432 subsp. aureus MW2] ZP_01826570.1 alcohol dehydrogenase,zinc-containing 65 4.00E−119 432 [Streptococcus pneumoniae SP14-BS69]CBW35926.1 alcohol dehydrogenase [Streptococcus pneumoniae 65 4.00E−119432 INV104] YP_003305918.1 Alcohol dehydrogenase zinc-binding domainprotein 63 4.00E−119 432 [Streptobacillus moniliformis DSM 12112]YP_003445415.1 alcohol dehydrogenase, propanol-preferring, 64 4.00E−119432 COG1064 [Streptococcus mitis B6] ZP_05900148.1 alcoholdehydrogenase, propanol-preferring 64 6.00E−119 431 [Leptotrichiahofstadii F0254] ZP_06901123.1 alcohol dehydrogenase [Streptococcusparasanguinis 64 1.00E−118 430 ATCC 15912] ZP_05685696.1 alcoholdehydrogenase [Staphylococcus aureus 62 1.00E−118 430 A9635]ZP_07728047.1 alcohol dehydrogenase, propanol-preferring 63 2.00E−118430 [Streptococcus parasanguinis F0405] ZP_04783075.1 alcoholdehydrogenase [Weissella paramesenteroides 62 4.00E−118 429 ATCC 33313]YP_003163500.1 Alcohol dehydrogenase GroES domain protein 63 7.00E−118427 [Leptotrichia buccalis DSM 1135] ZP_05745418.1 alcohol dehydrogenase[Lactobacillus antri DSM 63 3.00E−117 426 16041] ZP_07729093.1 alcoholdehydrogenase, propanol-preferring 63 4.00E−117 425 [Lactobacillus orisPB013-T2-3] ZP_03960690.1 alcohol dehydrogenase [Lactobacillus vaginalisATCC 61 1.00E−116 424 49540] YP_003920444.1 RBAM017440 [Bacillusamyloliquefaciens DSM7] 60 2.00E−116 422 ZP_04603652.1 hypotheticalprotein GCWU000324_03153 [Kingella 62 3.00E−116 422 oralis ATCC 51147]ZP_05553195.1 mycothiol-dependent formaldehyde dehydrogenase 634.00E−116 422 [Lactobacillus coleohominis 101-4-CHN] ZP_07073134.1alcohol dehydrogenase, propanol-preferring [Rothia 63 1.00E−115 421dentocariosa M567]

The foregoing detailed description has been given for clearness ofunderstanding only and no unnecessary limitations should be understoodthere from as modifications will be obvious to those skilled in the art.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

The disclosures, including the claims, figures and/or drawings, of eachand every patent, patent application, and publication cited herein arehereby incorporated herein by reference in their entireties.

What is claimed is:
 1. A recombinant microorganism comprising abiosynthetic pathway which uses a 3-keto acid as an intermediate,wherein said recombinant microorganism is engineered to reduce oreliminate the expression or activity of one or more enzymes catalyzingthe conversion of said 3-keto acid to a 3-hydroxyacid by-product.
 2. Therecombinant microorganism of claim 1, wherein said 3-keto acidintermediate is acetolactate and said 3-hydroxyacid by-product is2,3-dihydroxy-2-methylbutanoic acid (DH2MB).
 3. The recombinantmicroorganism of claim 2, wherein said recombinant microorganismproduces an acetolactate-derived product.
 4. The recombinantmicroorganism of claim 3, wherein said acetolactate-derived product isselected from isobutanol, 2-butanol, 1-butanol, 2-butanone,2,3-butanediol, acetoin, diacetyl, valine, leucine, pantothenic acid,isobutylene, 3-methyl-1-butanol, 4-methyl-1-pentanol, and coenzyme A. 5.The recombinant microorganism of claim 1, wherein said 3-keto acidintermediate is 2-aceto-2-hydroxybutyrate and said 3-hydroxyacidby-product is 2-ethyl-2,3-dihydroyxbutanoate.
 6. The recombinantmicroorganism of claim 5, wherein said recombinant microorganismproduces a 2-aceto-2-hydroxybutyrate-derived product.
 7. The recombinantmicroorganism of claim 6, wherein said 2-aceto-2-hydroxybutyrate-derivedproduct is selected from 2-methyl-1-butanol, isoleucine,3-methyl-1-pentanol, 4-methyl-1-hexanol, and 5-methyl-1-heptanol.
 8. Therecombinant microorganism of claim 1, wherein said enzyme catalyzing theconversion of a 3-keto acid to a 3-hydroxyacid by-product is a 3-ketoacid reductase.
 9. The recombinant microorganism of claim 8, whereinsaid 3-keto acid reductase is the S. cerevisiae YMR226 (SEQ ID NO: 1) ora homolog or variant thereof.
 10. The recombinant microorganism of claim8, wherein said 3-keto acid reductase is selected from SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22,and SEQ ID NO: 23, or homologs or variants thereof.
 11. A recombinantmicroorganism comprising a biosynthetic pathway which uses an aldehydeas an intermediate, wherein said recombinant microorganism is engineeredto reduce or eliminate the expression or activity of one or more enzymescatalyzing the conversion of said aldehyde to an acid by-product. 12.The recombinant microorganism of claim 11, wherein said biosyntheticpathway which uses an aldehyde as intermediate is selected from apathway for the biosynthesis of isobutanol, 1-butanol,2-methyl-1-butanol, 3-methyl-1-butanol, 1-propanol, 1-pentanol,1-hexanol, 3-methyl-1-pentanol, 4-methyl-1-pentanol, 4-methyl-1-hexanol,and 5-methyl-1-heptanol.
 13. The recombinant microorganism of claim 11,wherein said enzyme catalyzing the conversion of an aldehyde to an acidby-product is an aldehyde dehydrogenase.
 14. The recombinantmicroorganism of claim 13, wherein said aldehyde dehydrogenase is the S.cerevisiae ALD6 (SEQ ID NO: 25) or a homolog or variant thereof.
 15. Therecombinant microorganism of claim 13, wherein said aldehydedehydrogenase is selected from SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO:28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ IDNO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, or homologsor variants thereof.
 16. A recombinant microorganism comprising abiosynthetic pathway which uses a 3-keto acid and an aldehyde as anintermediate, wherein said recombinant microorganism is: (i) engineeredto reduce or eliminate the expression or activity of one or more enzymescatalyzing the conversion of said 3-keto acid to a 3-hydroxyacidby-product; and (ii) engineered to reduce or eliminate the expression oractivity of one or more enzymes catalyzing the conversion of saidaldehyde to an acid by-product.
 17. The recombinant microorganism ofclaim 16, wherein said enzyme catalyzing the conversion of a 3-keto acidto a 3-hydroxyacid by-product is a 3-keto acid reductase.
 18. Therecombinant microorganism of claim 17, wherein said 3-keto acidreductase is the S. cerevisiae YMR226 (SEQ ID NO: 1) or a homolog orvariant thereof.
 19. The recombinant microorganism of claim 17, whereinsaid 3-keto acid reductase is selected from SEQ ID NO: 2, SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8,SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ IDNO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, andSEQ ID NO: 23, or homologs or variants thereof.
 20. The recombinantmicroorganism of claim 16, wherein said enzyme catalyzing the conversionof an aldehyde to an acid by-product is an aldehyde dehydrogenase.